Small molecule-dependent inteins and uses thereof

ABSTRACT

Elucidating the function of proteins in mammalian cells is particularly challenging due to the inherent complexity of these systems. Methods to study protein function in living cells ideally perturb the activity of only the protein of interest but otherwise maintain the natural state of the host cell or organism. Ligand-dependent inteins offer single-protein specificity and other desirable features as an approach to control protein function in cells post-translationally. Some aspects of this invention provide second-generation ligand-dependent inteins that splice to substantially higher yields and with faster kinetics in the presence of the cell-permeable small molecule 4-HT, especially at 37° C., while exhibiting comparable or improved low levels of back-ground splicing in the absence of 4-HT, as compared to the parental inteins. These improvements were observed in four protein contexts tested in mammalian cells at 37° C., as well as in yeast cells assayed at 30° C. or 37° C. The newly evolved inteins described herein are therefore promising tools as conditional modulators of protein structure and function in yeast and mammalian cells.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application, U.S. Ser. No. 61/452,020, filed Mar. 12,2011, entitled “Small Molecule-Dependent Inteins and Uses Thereof,” theentire contents of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under grant R01GM065400, awarded by the National Institutes of Health (NIH), and grantHR0011-08-1-0085, awarded by the Defense Advanced Research ProjectsAgency (DARPA). The U.S. government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Methods to control protein structure and function inside living cellshave proven to be valuable tools to elucidate the roles of proteins intheir native biological contexts (Schreiber, 2003; Buskirk and Liu,2005; Banaszynski and Wandless, 2006). Traditional genetic methods thathave been widely used to control protein function by altering expressionlevels in mammalian cells include knock-out and knock-in systems such asthose mediated by Cre-Lox recombination (Sauer et al., 1988) and the useof transcriptional regulators such as the tetracycline-responsivetet-on/tet-off systems (Gossen et al., 1992). These methods are highlyspecific to the protein of interest and can be applied to many proteins,but typically require days to reach steady-state protein levels inmammalian cells, are irreversible in the case of recombination-basedmethods, and are vulnerable to transcriptional compensation(Shogren-Knaak et al., 2001; Marschang et al., 2004; Wong and Roth,2005; Acar et al., 2010). Other methods such as RNA interference (Fireet al., 1998), chemical genetics (Kino et al., 1987), small-moleculeregulated protein stability or degradation (Stankunas et al., 2003;Schneekloth et al., 2004; Banaszynski et al., 2006), and small moleculeinduced proteolytic shunts (Pratt et al., 2007) have also been usedeffectively by many researchers and offer more rapid control overprotein levels than strategies that exert control before transcription,but can require the discovery of small molecule modulators of proteinfunction, necessitate the involvement of other cellular machinery thatmay not be present in the cells of interest, or are prone to off-targeteffects.

Protein-splicing elements, termed inteins, can mediate profound changesin the structure and function of proteins. Inteins are analogous to theintrons found in polynucleotides. During intein-mediated proteinsplicing, inteins catalyze both their own excision from within apolypeptide chain and the ligation of the flanking external sequences(exteins), resulting in the formation of the mature protein from theexteins, and the free intein. No natural inteins, however, have beenshown to be regulated by small molecules. Extein function is typicallydisrupted by the presence of an intein but restored after proteinsplicing. Many inteins can splice in foreign extein environments.Therefore, inteins are powerful starting points for the creation ofartificial molecular switches.

Ligand-dependent inteins have been engineered (see, e.g., PCTapplication WO 2005/098043). Since inteins function in a variety ofextein environment, ligand-dependent inteins are universally applicableto regulate the activity of a variety of target proteins in mammaliancells in a ligand-dependent manner without disturbing transcriptional ortranslational pathways. However, conventional ligand-dependent inteinswere developed for use at room temperature and exhibit poor splicingefficiency or high background splicing in the absence of ligand whenincubated at higher temperatures. These characteristics limit theapplication of ligand-dependent inteins in mammalian cells.

SUMMARY OF THE INVENTION

Small-molecule-dependent inteins enable protein structure and functionto be controlled post-translationally in living cells. Previously, twointeins were evolved (2-4 and 3-2) that splice efficiently in thepresence, but not the absence, of the cell-permeable small molecule4-hydroxytamoxifen (4-HT) in a variety of extein contexts inSaccharomyces cerevisiae, as described in detail in International PCTPatent Application Serial Number PCT/US2005/010805, filed Mar. 30, 2005;U.S. Pat. No. 7,192,739, issued Mar. 20, 2007; and U.S. Pat. No.7,541,450, issued Jun. 2, 2009; the entire contents of each of which areincorporated by reference herein. In mammalian cells, however, the 2-4and 3-2 inteins exhibited significantly lower splicing efficiencies andslower splicing in the presence of 4-HT, as well as higher backgroundsplicing in the absence of 4-HT, than in yeast cells. These inteins aredescribed in detail in International PCT Patent Application SerialNumber PCT/US2005/010805, filed Mar. 30, 2005; U.S. Pat. No. 7,192,739,issued Mar. 20, 2007; and U.S. Pat. No. 7,541,450, issued Jun. 2, 2009;the entire contents of each of which are incorporated by referenceherein.

This invention relates to the development of improved intein variantsthat can splice efficiently, rapidly, and/or in a ligand-dependentmanner at about 37° C., for example, in cells of higher eukaryotes(e.g., mammalian cells). Results of new directed evolution efforts toimprove the splicing characteristics of 4-HT dependent inteins for useat about 37° C. and in mammalian cells are described herein. Theresulting second-generation inteins in yeast cells exhibit substantiallyimproved splicing activity and speed with no significant increase inbackground splicing at both 30° C. and 37° C. These second-generationinteins also splice with much greater speed and efficiency in mammaliancells, for example, in human cells, at 37° C. in four different exteincontexts compared with the parental inteins. These new ligand-dependentinteins represent more effective and broadly applicable tools for thesmall-molecule triggered, post-translational modulation of proteinactivities in living systems including mammalian cells.

In one aspect, this invention provides ligand-dependent inteins andintein domains that are optimized for applications in cells, tissues,and organisms that require incubation at temperatures in the range ofabout 30° C. to about 42° C., for example, at about 30° C., at about 35°C., at about 37° C., at about 37.5° C., at about 38° C., at about 38.5°C. at about 39° C., at about 39.5° C., or at about 40° C. In one aspect,this invention provides ligand-dependent inteins and intein domains thatare optimized for applications in mammalian cells, tissues, andorganisms, for example, in mouse or human cells, tissues, and organisms.In one aspect, this invention provides ligand-dependent inteins andintein domains that were evolved from the 2-4 and 3-2 inteins throughseveral additional rounds of mutation, recombination, and screening inS. cerevisiae at both 30° C. and 37° C. The resulting second-generationevolved inteins described herein exhibit substantially improved (˜2- to5-fold higher) splicing yields in yeast compared to the parental 2-4 and3-2 inteins and significantly faster splicing kinetics. The improvedproperties of these evolved inteins carried over to mammalian cells, inwhich the newly evolved inteins spliced with substantially greater (˜2-to 8-fold) efficiency in the presence of 4-HT while maintainingbackground splicing levels in the absence of 4-HT that are comparable toor better than the levels observed with the 2-4 or 3-2 inteins. Thesecond-generation evolved inteins augment the promise ofligand-dependent protein splicing as an effective and broadly applicableapproach to probing protein function in mammalian cells.

In one aspect, this invention provides methods for the use ofligand-dependent inteins. In some embodiments, methods for thegeneration of a hybrid protein comprising a ligand-dependent inteinprovided herein embedded into the amino acid sequence of a targetprotein are provided. In some embodiments, methods for the regulation oftarget protein activity via ligand-dependent inteins provided herein areprovided.

Other advantages, features, and uses of the invention will be apparentfrom the detailed description of certain non-limiting embodiments, thedrawings, which are schematic and not intended to be drawn to scale, andthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Intein evolution approach. (A) Overview of the directedevolution strategy used to isolate improved small molecule-dependentinteins. (B) Each round of evolution consisted of mutagenesis followedby at least two positive FACS screens in the presence of 1 μM 4-HT andone negative FACS screen in the absence of 4-HT. One set of FACS datafrom the Round 1 positive and negative screens is shown. (C) Two inteinevolution efforts were performed in parallel at 30° C. and 37° C.,comprising 20 total screening steps.

FIG. 2. Characterization of newly evolved inteins in yeast cells. (A)Representative Western blot of lysates from yeast cells expressingevolved intein variants in the context of Green Fluorescent Protein(GFP). Each lane shows lysate from 2.5×10⁶ cells after six hours ofgrowth at 30° C. in the absence or presence of 1 μM 4-HT visualized withan anti-FLAG-tag antibody. Quantitation of spliced and unspliced proteinbands by densitometry was used to calculate the percent spliced proteinshown in the rest of the figure. (B)-(E) Intein splicing characteristicsin yeast at various time points in the context of GFP at 30° C. ((D) and(E)) or 37° C. ((B) and (C)), either with ((B) and (D)) or without ((C)and (E)) 1 μM 4-HT. Error bars represent the standard deviation of atleast three independent experiments.

FIG. 3. Characterization of newly evolved inteins in mammalian cells.(A) Representative Western blot of lysates from HEK293 cells, a humanembryonic kidney-derived cell line, expressing evolved intein variantsin the context of GFP. Each lane shows lysate from cells after 12 hoursof growth at 37° C. in the absence or presence of 1 μM 4-HT processedwith an anti-FLAG-tag antibody to visualize spliced and unspliced GFP,and an anti-β-actin antibody to visualize β-actin, which served as aloading control. Quantitation of spliced and unspliced protein bands bydensitometry was used to calculate the percent spliced protein shown inthe rest of the figure. (B) and (C) Splicing characteristics of inteinsin the GFP context in HEK293 cells at 37° C. after 12 and 24 hoursincubation in the presence or absence of 1 μM 4-HT. Three evolvedinteins from the 37° C. evolution effort are shown in (B), and threeevolved inteins from the 30° C. evolution effort are shown in (C). Errorbars represent the standard deviation of at least three independentexperiments.

FIG. 4. Splicing characteristics of the 30R3-1 and 37R3-2 evolvedinteins in mammalian cells in three different protein contexts. HEK293cells expressing the inteins shown in the context of mCherry (A), Gli 1(B), or Gli3T (C) were incubated for 12 h or 24 h in the presence orabsence of 1 μM 4-HT. Unspliced and spliced protein was quantitated asdescribed in FIG. 3. Error bars represent the standard deviation of atleast three independent experiments.

FIG. 5. Reversion mutant analysis of evolved inteins 30R3-1 and 37R3-2.Each mutation in 30R3-1 ((A) and (B)) and 37R3-2 ((C) and (D)) relativeto the original 3-2 intein was reverted separately and the resultingintein variants in the context of GFP were characterized in yeast cellsat 30° C. ((A) and (C)) and at 37° C. ((B) and (D)). Yeast cell lysateswere prepared and analyzed by Western blot and densitometry after 6hours as described in FIG. 2. Error bars represent the standarddeviation of at least three independent experiments.

FIG. 6. Characterization of newly evolved inteins in yeast cells by flowcytometry. The P2 analysis gate indicates the cell population that ispositive for GFP fluorescence. (A) Representative FACS plots of cellstransformed with 30R3-1 intein in the GFP context treated with 1 μM 4-HTfor the durations shown at 30° C. and 37° C. The increase in cellfluorescence over time indicates an accumulation of functional, splicedGFP in cells treated with 4-HT. (B) Representative FACS plots of cellstransformed with 30R3-1 intein in the GFP context without 4-HT,incubated for 24 h. The lack of significant increase in the fluorescentpopulation over the course of 24 h indicates low levels of backgroundsplicing.

FIG. 7. Characterization of newly evolved inteins in mammalian cells byflow cytometry. Representative FACS plots of HEK293 cells transfectedwith DNA expressing the 37R3-2 intein in the GFP context without 4-HT ortreated with 1 μM 4-HT for 12 h or 24 h at 37° C. are shown.

FIG. 8. Characterization of the 30R3-1 and 37R3-2 evolved inteins in themCherry context in mammalian cells by flow cytometry. RepresentativeFACS plots of HEK293 cells transfected with DNA expressing the 2-4, 3-2,30R3-1, or 37R3-2 inteins in the context of mCherry, without 4-HT ortreated with 1 μM 4-HT for 24 h at 37° C. are shown. The P3 analysisgate indicates the cell population that is positive for mCherryfluorescence.

DEFINITIONS

As used herein and in the claims, the singular forms “a,” “an,” and“the” include the singular and the plural reference unless the contextclearly indicates otherwise. Thus, for example, a reference to “anagent” includes a single agent and a plurality of such agents.

The term “effective amount,” as used herein, refers to an amount of abiologically active agent that is sufficient to elicit a desiredbiological response. For example, in some embodiments, an effectiveamount of 4-HT may refer to the amount of 4-HT that inducesself-excision of a 4-HT-dependent intein from a hybrid protein. As willbe appreciated by the skilled artisan, the effective amount of a smallmolecule (e.g., 4-HT), a hybrid protein, or a polynucleotide, varydepending on various factors as, for example, on the desired biologicalresponse, the cells or tissues being targeted, the agent being used, andthe nature of the hybrid protein.

The term “extein,” as used herein, refers to an intein-flankingpolypeptide sequence that is ligated to another extein during theprocess of protein splicing to form a mature, spliced protein.Typically, an intein is flanked by two extein sequences that are ligatedtogether when the intein catalyzes its own excision. Exteins,accordingly, are the protein analog to exons found in mRNA. For example,a polypeptide comprising an intein may exhibit the structureextein(N)—intein—extein(C). After excision of the intein and splicing ofthe two exteins, the resulting structures are extein(N)—extein(C) and afree intein.

The term “hybrid protein,” as used herein, refers to a protein thatcomprises the amino acid sequence of a target protein and, embedded inthat amino acid sequence, a ligand-dependent intein as provided herein.Accordingly, a hybrid protein generally comprises the structure targetprotein(N)—intein—target protein(C). In some embodiments, a hybridprotein is encoded by a recombinant nucleic acid, in which a nucleicacid sequence encoding an intein is inserted in frame into a nucleicacid sequence encoding a target protein. In certain embodiments, thetarget protein exhibits a desired activity or property that is absent orreduced in the hybrid protein. In some embodiments, excision of theintein from the hybrid protein results in a restoration of the desiredactivity or property in the mature, spliced target protein. Non-limitingexamples of desired activities or properties of target proteins arebinding activities, enzymatic activities, reporter activities (e.g.,fluorescent activity), therapeutic activity, size, charge,hydrophobicity, hydrophilicity, or 3D-structure. In some embodiments,excision of the intein from a hybrid protein results in a mature,spliced target protein that exhibits the same or similar levels of adesired activity as the native target protein. A hybrid protein may becreated from any target protein by embedding an intein sequence into theamino acid sequence of the target protein, for example, by generating arecombinant, hybrid protein-encoding nucleic acid molecule andsubsequent transcription and translation, or by protein synthesismethods known to those of skill in the art.

The term “intein,” as used herein, refers to an amino acid sequence thatis able to excise itself from a protein and to rejoin the remainingprotein segments (the exteins) with a peptide bond in a process termedprotein splicing. Inteins are analogous to the introns found in mRNA.Many naturally occurring and engineered inteins and hybrid proteinscomprising such inteins are known to those of skill in the art and themechanism of protein splicing has been the subject of extensiveresearch. As a result, methods for the generation of hybrid proteinsfrom naturally occurring and engineered inteins are well known to theskilled artisan. For an overview, see pages 1-10, 193-207, 211-229,233-252, and 325-341 of Gross, Belfort, Derbyshire, Stoddard, and Wood(Eds.) Homing Endonucleases and Inteins Springer Verlag Heidelberg, ISBN9783540251064; the contents of which are incorporated herein byreference for disclosure of inteins and methods of generating hybridproteins comprising natural or engineered inteins. As will be apparentto those of skill in the art, an intein may catalyze protein splicing ina variety of extein contexts. Accordingly, an intein can be introducedinto virtually any target protein sequence to create a desired hybridprotein, and the invention is not limited in the choice of targetproteins.

The term “intein domain,” as used herein, refers to the amino acidsequence of an intein that is essential for self-excision and exteinligation. For example, in some inteins, the entire intein amino acidsequence, or part(s) thereof, may constitute the intein domain, while inligand-dependent inteins, the ligand-binding domain is typicallyembedded into the intein domain, resulting in the structure inteindomain (N)—ligand-binding domain—intein domain (C).

The term “ligand binding domain,” as used herein, refers to a peptide orprotein domain that binds a ligand. A ligand binding domain may be anaturally occurring or an engineered domain. Examples of ligand-bindingdomains referred to herein are the ligand binding domain of a nativeestrogen receptor, e.g., the ligand-binding domain of the native humanestrogen receptor, and engineered, evolved, or mutated derivativesthereof. Typically, a ligand-binding domain useful in the context ofligand-dependent inteins, as provided herein, exhibits a specificthree-dimensional structure in the absence of the ligand, which inhibitsintein self-excision, and undergoes a conformational change upon bindingof the ligand, which promotes intein self-excision. Some of theligand-dependent inteins provided herein comprise a ligand-bindingdomain derived from the estrogen receptor that can bind 4-HT and otherestrogen-receptor ligands, e.g., ligands described in more detailelsewhere herein, and undergo a conformational change upon binding ofthe ligand. An appropriate ligand may be any chemical compound thatbinds the ligand-binding domain and induces a desired conformationalchange. In some embodiments, an appropriate ligand is a molecule that isbound by the ligand-binding domain with high specificity and affinity.In some embodiments, the ligand is a small molecule. In someembodiments, the ligand is a molecule that does not naturally occur inthe context (e.g., in a cell or tissue) that a ligand-dependent inteinis used in. For example, in some embodiments, the ligand-binding domainis a ligand-binding domain derived from an estrogen receptor, and theligand is tamoxifen or a derivative or analog thereof (e.g.,hydroxytamoxifen, 4-HT).

The term “ligand-dependent intein,” as used herein refers to an inteinthat comprises a ligand-binding domain. Typically, the ligand-bindingdomain is inserted into the amino acid sequence of the intein, resultingin a structure intein (N)—ligand-binding domain—intein (C). Typically,ligand-dependent inteins exhibit no or only minimal protein splicingactivity in the absence of an appropriate ligand, and a marked increaseof protein splicing activity in the presence of the ligand. In someembodiments, the ligand-dependent intein does not exhibit observablesplicing activity in the absence of ligand but does exhibit splicingactivity in the presence of the ligand. In some embodiments, theligand-dependent intein exhibits an observable protein splicing activityin the absence of the ligand, and a protein splicing activity in thepresence of an appropriate ligand that is at least 5 times, at least 10times, at least 50 times, at least 100 times, at least 150 times, atleast 200 times, at least 250 times, at least 500 times, at least 1000times, at least 1500 times, at least 2000 times, at least 2500 times, atleast 5000 times, at least 10000 times, at least 20000 times, at least25000 times, at least 50000 times, at least 100000 times, at least500000 times, or at least 1000000 times greater than the activityobserved in the absence of the ligand. In some embodiments, the increasein activity is dose dependent over at least 1 order of magnitude, atleast 2 orders of magnitude, at least 3 orders of magnitude, at least 4orders of magnitude, or at least 5 orders of magnitude, allowing forfine-tuning of intein activity by adjusting the concentration of theligand.

The term “mutation,” as used herein, refers to an alteration, forexample, a deletion, substitution, addition, inversion, duplication, ormultiplication of a residue or a plurality of residues, in a sequence ofresidues, for example, in a nucleic acid or peptide sequence. Forexample, in some embodiments, the term mutation refers to a substitutionof an amino acid residue of a protein, e.g., a ligand-dependent intein,with a different amino acid residue. In some embodiments, the termmutation refers to a substitution of a nucleotide residue of a nucleicacid molecule, e.g., a nucleic acid molecule encoding a ligand-dependentintein, with a different nucleotide. In some such embodiments, themutation in the intein-encoding nucleic acid results in a substitutionof an amino acid in the encoded protein.

The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide”are used interchangeably herein, and refer to a polymer ofribonucleotides (RNA molecules) or deoxyribonucleotides (DNA molecules)in either single-stranded, or double-stranded form. Double-strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule refers to the primary and secondary structure of the molecule,and does not limit it to any particular tertiary forms. Thus, these terminclude, for example, double-stranded DNA found in linear (e.g.,restriction fragments) or circular DNA molecules, plasmids, andchromosomes. Nucleotide sequences of nucleic acid molecules aredescribed in 5′-to-3′ direction.

The terms “peptide” and “protein” are used interchangeably herein andrefer to a molecule that comprises a polymer of at least three aminoacids linked together by peptide (amide) bonds. Peptides can comprisenatural amino acids, non-natural amino acids, and/or amino acid analogs.A peptide may comprise an amino acid that is modified, for example, bythe addition of a chemical entity such as a carbohydrate group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification (e.g., amidation). In some embodiments, a peptidecomprising an amino acid modification exhibits an increased stability orbiological activity as compared to its unmodified counterpart. Peptidesequences are given by convention starting with the amino-terminus(N-terminus, N) and ending with the carboxy-terminus (C-terminus, C).

The term “protein splicing,” as used herein, refers to a process inwhich a sequence, an intein, is excised from within an amino acidsequence, and the remaining fragments of the amino acid sequence, theexteins, are ligated via an amide bond to form a continuous amino acidsequence.

The term “small molecule,” as used herein, refers to a non-peptidic,non-oligomeric organic compound either prepared in the laboratory orfound in nature. Small molecules, as used herein, can refer to compoundsthat are “natural product-like”, however, the term “small molecule” isnot limited to “natural product-like” compounds. Rather, a smallmolecule is typically a non-polymeric, non-oligomeric molecule that ischaracterized in that it contains several carbon-carbon bonds, and has amolecular weight of less than 2000 g/mol, preferably less than 1500g/mol, although this characterization is not intended to be limiting forthe purposes of the present invention.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Protein splicing elements known as inteins are able to catalyze theirexcision out of a single polypeptide and leave behind the flankingsequences, or exteins, precisely ligated together through a nativepeptide bond (Paulus, 2000). Inteins are attractive tools for modulatingprotein expression because they do not require any other cellularcomponents, are able to splice out of a wide variety of extein contexts(Xu et al., 1993), and can undergo splicing in minutes (Paulus, 2000).Although natural inteins splice spontaneously, inteins that undergosplicing in a small molecule-dependent manner have been developed byfusing intein halves with proteins that dimerize in the presence of asmall molecule (Mootz and Muir, 2002; Mootz et al., 2003; Shi and Muir,2005), or by directed evolution in which a library of intact inteinsfused to a ligand-binding domain was screened to splice in the presence,but not the absence, of a small molecule (Buskirk et al., 2004). Thesesmall molecule-dependent inteins have enabled protein function in cellsto be controlled post-translationally by the addition of an exogenous,cell-permeable molecule (Mootz and Muir, 2002, Mootz et al., 2003,Buskirk et al., 2004; Mootz et al., 2004; Shi and Muir, 2005; Yuen etal., 2006; Schwartz et al., 2007; Hartley and Madhani, 2009). Forexample, in some embodiments, a hybrid protein comprising aligand-dependent intein embedded into the amino acid sequence of atarget protein is expressed in a cell, for example, a human cell, in theabsence of an appropriate ligand. If the intein disrupts the activity ofthe target protein, and no native target protein is present in the cell,then there is no target protein present in the cell. In someembodiments, the cell is contacted with an appropriate ligand activatingthe ligand-dependent intein. As a result, the intein self-excises fromthe hybrid protein, generating a mature, spliced target protein. In someembodiments, this protein splicing restores the activity of the targetprotein. To give but one example, if the target protein is a fluorescentprotein, cells expressing the hybrid protein in the absence of ligandare non-fluorescent, while fluorescence can be observed in cellscontacted with ligand.

Previously, variants of the Mycobacterium tuberculosis RecA intein weredeveloped that selectively splice in the presence of the cell-permeablesmall molecule 4-hydroxytamoxifen (4-HT) in a rapid, dose-dependentmanner using directed evolution in S. cerevisiae (Buskirk et al., 2004).The M. tuberculosis RecA intein was chosen because it can efficientlysplice in a wide variety of contexts (Lew and Paulus, 2002), and theevolved 4-HT-triggered inteins retained this characteristic. Theseevolved inteins have been successfully used as a tool to study the roleof histone H2A.Z in establishing chromatin architecture around promoterregions in S. cerevisiae (Hartley and Madhani, 2009). It will beappreciated by those of skill in the art that other inteins can also beevolved according to methods described herein and the invention is notlimited in this respect.

It was demonstrated that these evolved inteins are functional inmammalian cells at 37° C. but splice with significantly reduced speed,lower efficiency, and higher background splicing in the absence of 4-HTcompared with splicing at 30° C. in yeast (Yuen et al., 2006). Theselimitations constrain the utility of these evolved inteins as tools formammalian cell biology; indeed, only two studies (Mootz et al., 2003;Yuen et al., 2006) have reported the use of small molecule-dependentinteins in mammalian cells.

This invention relates to the development of improved ligand-dependentintein and intein domain variants that can splice efficiently, rapidly,and/or in a ligand-dependent manner at about 37° C., for example, incells of higher eukaryotes (e.g., mammalian cells). Results of newdirected evolution efforts to improve the splicing characteristics of4-HT dependent inteins for use at about 37° C. and in mammalian cellsare described herein. The resulting second-generation inteins in yeastcells exhibit substantially improved splicing activity and speed with nosignificant increase in background splicing at both 30° C. and 37° C.These second-generation inteins also splice with much greater speed andefficiency in mammalian cells, for example, in human cells, at 37° C. infour different extein contexts compared with the parental inteins. Thesenew ligand-dependent inteins represent more effective and broadlyapplicable tools for the small-molecule triggered, post-translationalmodulation of protein activities in living systems including mammaliancells.

Ligand-Dependent Inteins

Inteins are polypeptide sequences embedded within a protein. Inteinscatalyze their own excision from the peptide chain and ligation of theresulting ends of the protein. The self-excision catalyzed by the inteinresults in a mature, spliced protein and a free intein. Whilenaturally-occurring inteins catalyze protein splicing in a spontaneousmanner, the splicing activity of the inteins provided herein isdependent on a ligand, for example, a small molecule ligand.

The ligand-dependent inteins provided herein comprise a modifiedligand-binding domain of the estrogen receptor protein, embedded into amodified RecA intein from M. tuberculosis. In some embodiments, theligand-binding domain is derived from the an estrogen receptor protein,for example, from the human estrogen receptor. The sequence of the humanestrogen receptor and the location of the ligand-binding domain withinthe human estrogen receptor protein are well known to those of skill inthe art. Non-limiting, exemplary sequences of the human estrogenreceptor can be retrieved from RefSeq database entries NP_000116(isoform 1); NP_001116212 (isoform 2); NP_001116213 (isoform 3); andNP_001116214 (isoform 4) from the National Center for BiotechnologyInformation (NCBI, www.ncbi.nlm.nih.gov). In some embodiments, theligand-binding domain of a ligand-dependent intein provided herein isderived from a sequence comprising amino acid residues 304-551 of thehuman estrogen receptor.

It will be appreciated by those of skill in the art that other ligandbinding domains are also useful in connection with the intein domainsdescribed herein. For example, some aspects of this invention provideligand-dependent inteins that comprise an N-terminal and a C-terminalintein domain as described herein, and a central ligand-binding domain,for example, a ligand-binding domain of a hormone-binding protein, e.g.,of an androgen receptor, an estrogen receptor, an ecdysone receptor, aglucocorticoid receptor, a mineralocorticoid receptor, a progesteronereceptor, a retinoic acid receptor, or a thyroid hormone receptorprotein. Ligand-binding domains of hormone-binding receptors, induciblefusion proteins comprising such ligand-binding domains, and methods forthe generation of such fusion proteins are well known to those of skillin the art (see, e.g., Becker, D., Hollenberg, S., and Ricciardi, R.(1989). Fusion of adenovirus E1A to the glucocorticoid receptor byhigh-resolution deletion cloning creates a hormonally inducible viraltransactivator. Mol. Cell. Biol. 9, 3878-3887; Boehmelt, G., Walker, A.,Kabrun, N., Mellitzer, G., Beug, H., Zenke, M., and Enrietto, P. J.(1992). Hormone-regulated v-rel estrogen receptor fusion protein:reversible induction of cell transformation and cellular geneexpression. EMBO J 11, 4641-4652; Braselmann, S., Graninger, P., andBusslinger, M. (1993). A selective transcriptional induction system formammalian cells based on Ga14-estrogen receptor fusion proteins. ProcNatl Acad Sci USA 90, 1657-1661; Furga G, Busslinger M (1992).Identification of Fos target genes by the use of selective inductionsystems. J. Cell Sci. Suppl 16,97-109; Christopherson, K. S., Mark, M.R., Bajaj, V., and Godowski, P. J. (1992). Ecdysteroid-dependentregulation of genes in mammalian cells by a Drosophila ecdysone receptorand chimeric transactivators. Proc Natl Acad Sci USA 89, 6314-8; Eilers,M., Picard, D., Yamamoto, K., and Bishop, J. (1989). Chimaeras of Myconcoprotein and steroid receptors cause hormone-dependent transformationof cells. Nature 340, 66-68; Fankhauser, C. P., Briand, P. A., andPicard, D. (1994). The hormone binding domain of the mineralocorticoidreceptor can regulate heterologous activities in cis. Biochem BiophysRes Commun 200, 195-201; Godowski, P. J., Picard, D., and Yamamoto, K.R. (1988). Signal transduction and transcriptional regulation byglucocorticoid receptor-LexA fusion proteins. Science 241, 812-816;Kellendonk, C., Tronche, F., Monaghan, A., Angrand, P., Stewart, F., andSchutz, G. (1996). Regulation of Cre recombinase activity by thesynthetic steroid RU486. Nuc. Acids Res. 24, 1404-1411; Lee, J. W.,Moore, D. D., and Heyman, R. A. (1994). A chimeric thyroid hormonereceptor constitutively bound to DNA requires retinoid X receptor forhormone-dependent transcriptional activation in yeast. Mol Endocrinol 8,1245-1252; No, D., Yao, T. P., and Evans, R. M. (1996).Ecdysone-inducible gene expression in mammalian cells and transgenicmice. Proc Natl Acad Sci USA 93, 3346-3351; and Smith, D., Mason, C.,Jones, E., and Old, R. (1994). Expression of a dominant negativeretinoic acid receptor g in Xenopus embryos leads to partial resistanceto retinoic acid. Roux's Arch. Dev. Biol. 203, 254-265; all of which areincorporated herein by reference in their entirety). Additionalligand-binding domains useful for the generation of ligand-dependentinteins as provided herein will be apparent to those of skill in the artand the invention is not limited in this respect.

The ligand-dependent inteins provided herein are inactive (or onlyminimally active) in the absence of the appropriate ligand, but can beinduced to be active, and, thus, to self-excise, by contacting them witha ligand that binds the ligand-binding domain of the human estrogenreceptor. Small molecule ligands binding the ligand-binding domain ofthe estrogen receptor (e.g., the human estrogen receptor), and thususeful to induce the activity of the ligand-dependent inteins describedherein, are well known to those of skill in the art. In someembodiments, the ligand used to induce the activity of theligand-dependent inteins described herein specifically binds to theligand-binding domain of the estrogen receptor. In some embodiments, theligand binds the ligand-binding domain of a ligand-dependent inteinprovided herein with high affinity, for example, with an affinity of atleast about 10⁻¹⁰ M, at least about 10⁻⁹ M, at least about 10⁻⁸ M, atleast about 10⁻⁷ M, at least about 10⁻⁶ M, or at least about 10⁻⁵ M.Examples of appropriate estrogen receptor-binding ligands that areuseful to induce the activity of the ligand-dependent inteins providedherein, for example, the ligand-dependent inteins provided in SEQ IDNOs: 3-8, include, but are not limited to, 17β-estradiol, 17α-ethynylestradiol, tamoxifen and tamoxifen analogs (e.g., 4-hydroxytamoxifen(4-HT, 4-OHT), 3-hydroxytamoxifen (droloxifene)), tamoxifen metabolites(e.g., hydroxytamoxifen, endoxifen), raloxifene, toremifene, ICI-182,and ICI-780. Other useful ligands will be apparent to those of skill inthe art, and the invention is not limited in this respect.

In some embodiments, the ligand-dependent intein is inactive or onlyminimally active in the absence of an appropriate ligand. In someembodiments, the self-excision activity of the ligand-dependent inteinis increased in the presence of an appropriate ligand. In someembodiments, the ligand increases the activity of a ligand-dependentintein provided herein in a concentration-dependent manner, with lowligand concentration levels translating to low intein activity levels,and high ligand concentration levels translating to high intein activitylevels. In some embodiments where intein activity is induced in livingcells, the concentration of the ligand and the time of exposure of thecells to the ligand are chosen to be non-toxic to the cells. Ligand maybe non-toxic over a whole range of concentrations.

In some embodiments, a ligand-dependent intein provided herein does notexhibit observable splicing activity in the absence of an appropriateligand, but does exhibit splicing activity in the presence of such aligand. In some embodiments, a ligand-dependent intein provided hereinexhibits an increase in splicing activity of at least about 5-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 25-fold, at least about 30-fold, at least about 40-fold, atleast about 50-fold, at least about 75-fold, at least about 100-fold, atleast about 150-fold, at least about 200-fold, at least about 250-fold,at least about 500-fold, at least about 1000-fold, at least about2000-fold, at least about 5000-fold, at least about 10000-fold, at leastabout 50000-fold, at least about 100000-fold, at least about500000-fold, or at least about 1000000 fold, in the presence of a ligandfor the ligand-binding domain, e.g., 4-HT, as compared to its baselineactivity in the absence of the ligand. In some embodiments, aligand-dependent intein provided herein exhibits a level of proteinsplicing activity in the presence of an appropriate ligand, e.g., 4-HT,that is similar to the splicing activity of the RecA intein observed orexpected under the same conditions. In some embodiments, aligand-dependent intein provided herein exhibits a splicing activity inthe presence of an appropriate ligand, e.g., 4-HT, that is greater thanthe splicing activity of the RecA intein observed or expected under thesame conditions, for example, by a factor of at least 2, at least 3, atleast, 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 20, at least 25, at least 30, at least 40, at least50, or at least 100.

Intein Domains

The invention provides intein domains for use in ligand-dependent inteinapplications at temperatures within the range of about 30° C. to about37° C., for example, in mammalian cells that are cultured at about 37°C. In some embodiments, the optimized intein domains provided hereincomprise an N-terminal intein domain and a C-terminal intein domain. Toobtain a ligand-dependent intein, the N- and C-terminal intein domainsare fused to a central ligand-binding domain, for example, to theligand-binding domain of a steroid (e.g., estrogen) receptor protein. Insome embodiments, the ligand-binding domain is a native ligand-bindingdomain, while in other embodiments, the ligand-binding domain is alsooptimized for use at temperatures within the range of about 30° C. toabout 37° C. For example, in some embodiments, the ligand-binding domainis optimized for use in mammalian cells that are cultured at about 37°C. In some embodiments, the optimized intein domains provided herein arederived from naturally-occurring intein domains by evolution methodsdescribed herein. In some embodiments, the optimized intein domainsprovided herein are derived from the RecA intein domain as described inmore detail herein.

In some embodiments, an intein domain is provided that comprises thesequence: CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAV*AKDGTLLARPVVSWFDQGTRDVIGLRIAGGAI*VWATPDHKVLTEYGWRAAGELRKGDRVARVQAFADALDDKFLHDMLAEE*LRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN (SEQ ID NO: 9), wherein atleast one of the amino acid residues followed by an asterisk (*) ismutated (e.g., substituted with a different amino acid residue ordeleted). In some embodiments, the optimized intein domain comprises aV*→A (Valine* to Alanine) mutation, an I*→T (Isoleucine* to Threonine)mutation, and/or an E*→G (Glutamate* to Glycine) mutation.Ligand-dependent inteins comprising a ligand-binding protein domain, forexample, a ligand-binding domain of the estrogen receptor, inserted intothe intein domain sequence provided above, are also provided.

In some embodiments, an N-terminal intein domain (intein-N) is providedthat comprises the amino acid sequence:CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAV*AKDGTLLARPVVSWFDQGTRDVIGLRIAGGAI*VWATPDHKVLTEYGWRAAGELRKGDRVA(SEQ ID NO: 10), wherein at least one of the amino acid residuesfollowed by an asterisk is mutated. In some embodiments, the N-terminalintein domain comprises a V*→A mutation and/or an I*→T mutation.

In some embodiments, an optimized C-terminal intein domain (intein-C) isprovided that comprises the amino acid sequence:RVQAFADALDDKFLHDMLAEE*LRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN (SEQ ID NO:11), wherein the E* residue is mutated. In some embodiments, themutation is an E*G mutation. In some embodiments, a ligand-dependentintein is provided that comprises the N-terminal intein domain and/orthe C-terminal intein domain described above, and a ligand-bindingdomain, for example, a ligand-binding domain of the steroid (e.g.,estrogen) receptor as described herein. In some embodiments, aligand-dependent intein is provided that comprises the N-terminal andthe C-terminal intein domains described above and a centralligand-binding domain. In some embodiments, the ligand-binding domain isa ligand-binding domain described herein, for example, theligand-binding domain of an estrogen receptor protein, or an optimized,mutated derivative thereof. In some embodiments, the ligand-bindingdomain comprises the amino acid sequence:NSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHA (SEQ ID NO: 12). In some embodiments, theligand-binding domain comprises the amino acid sequence:NSLALSLTADQMVSALLDAEPPIL*YSEYD*PTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLEC*AWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYT*NVVPLYDLLLEMLDAHRLHA (SEQ ID NO: 13), wherein at least one of theresidues L*, D*, C*, or T* is mutated. In some embodiments, theligand-binding domain comprises an L*→P (Leucine* to Proline), a D*→N(Aspartate* to Asparagine), a C*→R (Cysteine* to Arginine), and/or aT*→K ((Threonine* to Lysine) mutation.

Ligand-Binding Domain

The invention also provides ligand-binding protein domains. Theligand-binding domains provided herein are derived from the humanestrogen receptor ligand-binding domain and can be used to generateligand-dependent proteins, for example, ligand-dependent inteins. Usefulligands that bind to the ligand-binding domains provided herein aredescribed in more detail elsewhere herein. Non-limiting examples of suchligands include tamoxifen and tamoxifen analogs and derivatives (e.g.,4-HT).

Some embodiments provide a ligand-binding protein domain comprising theamino acid sequence:NSLALSLTADQMVSALLDAEPPIL*YSEYD*PTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLEC*AWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYT*NVVPLYDLLLEMLDAHRLHA (SEQ ID NO: 13). In some embodiments, at leastone of the residues L*, D*, C*, or T* is mutated. In some embodiments,the estrogen-binding domain comprises an L*P, a D*N, a C*R, and/or a T*Kmutation. In some embodiments, the ligand-binding domain comprises theligand-binding domain provided in any of SEQ ID NOs 3-8. Theligand-binding domain in SEQ ID NOs 3-8 can be identified by those ofskill in the art, for example, by sequence alignment of any of thesequences in SEQ ID NOs 3-8 with the ligand-dependent domain providedabove.

In some embodiments, a ligand-binding domain described herein is fusedto an N-terminal intein domain and a C-terminal intein domain, thusforming a ligand-dependent intein of the structureintein(N)—ligand-binding domain—intein(C). In some embodiments, theC-terminal and/or the N-terminal intein domain is a naturally occurringintein domain. Naturally occurring intein domains are well known tothose of skill in the art (e.g., as described in International PCTPatent Application, Serial Number PCT/US2005/010805, filed Mar. 30,2005; U.S. Pat. No. 7,192,739, issued Mar. 20, 2007; U.S. Pat. No.7,541,450, issued Jun. 2, 2009; and on pages 1-10, 193-207, 211-229,233-252, and 325-341 of Gross, Belfort, Derbyshire, Stoddard, and Wood(Eds.) Homing Endonucleases and Inteins Springer Verlag Heidelberg, ISBN9783540251064; the contents of each of which are incorporated herein byreference), and the invention is not limited in this respect.

In some embodiments, the ligand-binding domain is embedded in the inteinbetween the N-and the C-terminal intein domains. In other embodiments,the ligand-binding domain is embedded anywhere in the intein amino acidsequence.

In some embodiments, an ligand-binding domain described herein (e.g., ahuman estrogen receptor-derived ligand-binding domain) is fused to aRecA-derived intein domain, for example, an intein domain as describedherein. In some embodiments, an estrogen-binding domain describedherein, for example, the estrogen-binding domain provided in any of SEQID NOs 3-8 are fused to an N-terminal and a C-terminal intein domain asprovided herein (e.g., the intein domain provided in any of SEQ ID NOs:3-8).

In some embodiments, a ligand-binding domain, e.g., an estrogen-bindingdomain, provided herein is fused to an N-terminal and a C-terminalintein sequence as provided in any one of SEQ ID NOs: 3-8 to generate aligand-dependent intein of the structure intein(N)—ligand-bindingdomain—intein(C). For example, in such embodiments, the estrogen-bindingdomain of SEQ ID NO: 3 is fused to the N-terminal and the C-terminalintein domains of SEQ ID NO: 4. In some other such embodiments, theestrogen-binding domain of SEQ ID NO: 3 is fused to the N-terminal andthe C-terminal intein domains of SEQ ID NO: 5. In some other suchembodiments, the estrogen-binding domain of SEQ ID NO: 7 is fused to theN-terminal and the C-terminal intein domains of SEQ ID NO: 6, and so on.

In some embodiments, an estrogen-binding domain provided herein is fusedto an N-terminal intein domain as provided in any of SEQ ID NOs: 3-8,and to a C-terminal intein sequence as provided in any of SEQ ID NOs:3-8, wherein the N-terminal and the C-terminal intein domains are notfrom the same SEQ ID NO. For example, in some such embodiments, theestrogen-binding domain of SEQ ID NO: 5 is fused to the N-terminalintein of SEQ ID NO: 3 and to the C-terminal intein domains of SEQ IDNO: 4. In some other such embodiments, the estrogen-binding domain ofSEQ ID NO: 3 is fused to the N-terminal intein domain of SEQ ID NO: 6and the C-terminal intein domains of SEQ ID NO: 7. In some other suchembodiments, the estrogen-binding domain of SEQ ID NO: 7 is fused to theN-terminal intein domain of SEQ ID NO: 7 and the C-terminal inteindomains of SEQ ID NO: 3, and so on.

In some embodiments, a ligand-binding domain provided herein is fused toan N-terminal intein domain and a C-terminal intein domain, forming aligand-dependent intein. In some embodiments, the N-terminal inteindomain comprises the sequenceCLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAV*AKDGTLLARPVVSWFDQGTRDVIGLRIAGGAI*VWATPDHKVLTEYGWRAAGELRKGDRVA (SEQ ID NO: 10). In someembodiments, the N-terminal intein sequence comprises the sequence abovewith a mutation of the V* and/or the I* residue(s). In some embodiments,the N-terminal intein sequence comprises the sequence above with a V*→A,and/or an I*→T mutation. In some embodiments, the C-terminal inteindomain comprises the sequenceRVQAFADALDDKFLHDMLAEE*LRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HN (SEQ ID NO:11). In some embodiments, the C-terminal intein sequence comprises thesequence above with a mutation of the E* residue. In some embodiments,the C-terminal intein sequence comprises the sequence above with an E*→Gmutation.

Ligand-Dependent Inteins

The invention provides ligand-dependent inteins that are optimized foruse at temperatures within the range of about 30° C. to about 42° C.,for example, for use at about 37° C. Some of the optimized inteinsprovided herein comprise a RecA-derived intein domain and aligand-binding domain and are of the general structureintein(N)—ligand-binding domain—intein(C). Some of the optimized inteinsprovided herein are derived from the 3-2 intein as described in detailin International PCT Patent Application Serial Number PCT/US2005/010805,filed Mar. 30, 2005; U.S. Pat. No. 7,192,739, issued Mar. 20, 2007; andU.S. Pat. No. 7,541,450, issued Jun. 2, 2009; all of which are entitled“Ligand-dependent Protein Splicing,” and the entire contents of each ofwhich are incorporated herein by reference. The 3-2 intein was derivedfrom the 2-4 intein, also described in the above-referenced US patents.

The inteins provided herein are optimized for use at temperatures withinthe range of about 30° C. to about 37° C., for example, in mammaliancells that are cultured at about 37° C. The provided intein sequencesare universally applicable to regulate the activity of any targetprotein post-translationally, since they can be inserted into any targetprotein to generate an inactive hybrid protein that can be spliced in aligand-dependent manner to restore target protein activity, as describedin more detail elsewhere herein.

In some embodiments, the optimized intein sequence comprises the aminoacid sequence of the 30R3-1 intein (mutations as compared to the 3-2intein sequence are underlined):

(SEQ ID NO: 3) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC.

In some embodiments, an intein is provided that comprises or consists ofthe amino acid sequence of the 30R3-2 intein:

(SEQ ID NO: 4) CLAEGTRIFDPVTGTTHREEDVVDGRKPLHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC.

In some embodiments, an intein is provided that comprises or consists ofthe amino acid sequence of the 30R3-3 intein:

(SEQ ID NO: 5) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEEFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKTIDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC.

In some embodiments, an intein is provided that comprises or consists ofthe amino acid sequence of the 37R3-1 intein:

(SEQ ID NO: 6) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEASMMGLLTNLADRELVHMENWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSILKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC.

In some embodiments, an intein is provided that comprises or consists ofthe amino acid sequence of the 37R3-2 intein:

(SEQ ID NO: 7) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC.

In some embodiments, an intein is provided that comprises or consists ofthe amino acid sequence of the 37R3-3 intein:

(SEQ ID NO: 8) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPELYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC.

In some embodiments, a ligand-dependent intein is provided, wherein theintein does not comprise a sequence as provided in SEQ ID NO: 1 or SEQID NO:2, and wherein the intein comprises an amino acid sequence atleast 90%, at least 95%, at least 98%, or at least 99% identical to anyof the amino acid sequences provided in SEQ ID NOs 3-8. The level ofsequence identity can be determined by methods known to those of skillin the art, for example, by sequence alignment. In some embodiments, theterm at least 99% sequence identity refers to a level of identitybetween two sequences (e.g., amino acid sequences), in which at least 99out of 100 residues (e.g., amino acid residues) are identical, if thesequences are optimally aligned. The optimal alignment of two sequencesis typically the alignment in which a maximum number of residue identitymatches is observed between the two sequences.

Hybrid Protein

The ligand-dependent inteins provided herein have the ability to excisethemselves from a target protein they are embedded in, and to ligate theresulting ends of the protein together, resulting in a mature, splicedprotein and a free intein. A protein comprising a target proteinsequence and, integrated into this sequence, an intein sequence, isreferred to herein as a hybrid protein. In some embodiments, a hybridprotein is provided that comprises an intein or intein domain describedherein, for example, a ligand-dependent intein provided in any of SEQ IDNOs: 3-8. Typically, the ligand-dependent intein sequence is integratedinto a target protein sequence to form the hybrid protein structuretarget protein (N)—intein (N)—ligand binding domain—intein (C)—targetprotein (C). In some embodiments, the intein is inserted into anα-helical region of the target protein. In some embodiments, the inteinis inserted into a β-strand of the target protein. In some embodiments,in the presence of an appropriate ligand binding the ligand-dependentintein, the ligand dependent intein catalyzes the excision of theligand-dependent intein and ligation of the target protein forming themature, spliced target protein of the structure target (N)-target (C).In some embodiments, the hybrid protein does not exhibit a function ofthe native target protein. In some embodiments, a function of the targetprotein is disrupted or diminished by the inserted intein sequence. Insome embodiments, excision of the intein from the hybrid proteinrestores target protein function in the spliced, mature target protein.In some embodiments, the ligand is HT-4.

For example, in some embodiments, a ligand-dependent intein as providedherein is embedded in a reporter protein (target protein), for example,a fluorescent protein (e.g., GFP), and the resulting hybrid protein doesnot exhibit fluorescence or only minimal fluorescence as compared to thenative reporter protein. In some embodiments, the mature, splicedreporter protein, now devoid of the intein, exhibits similar or onlyslightly decreased reporter activity (e.g., fluorescence) as compared tothe native reporter protein.

In some embodiments, the function of the target protein is fullyrestored upon ligand-induced self-excision of the ligand-dependentintein from the target protein. In some embodiments, the function of thetarget protein is restored to a significant extent upon ligand-inducedself-excision of the ligand-dependent intein from the target protein.For example, in some embodiments, the mature, spliced target proteinexhibits at least about 80%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or at least about 100% of theactivity exhibited by the native target protein.

In some embodiments, the self-excision of a ligand-dependent inteinprovided herein leaves no “scar” or only a minimal “scar” in theresulting intein-free protein. For example, in some embodiments, thesplicing reaction either leaves no amino acid residue from the inteinsequence or just a short sequence of one, two, or three amino acidresidues. In certain embodiments, the splicing reaction leaves no aminoacid residues from the intein in the mature protein after self-excision.In other embodiments, the splicing reaction leaves one amino acidresidue from the intein, for example, a cysteine residue. In someembodiments, the amino acid residue(s) from the intein left in themature, spliced protein do(es) not interfere, or interfere(s) onlyminimally, with the activity of the mature, spliced protein, e.g., doesnot inhibit an activity of interest exhibited by the mature, splicedprotein as compared to the native target protein.

Target Proteins

The ligand-dependent inteins provided herein can be inserted into anytarget protein to create a hybrid protein of the structure targetprotein (N)—intein—target protein(C). Such engineered hybrid proteinscan be synthesized de novo by protein synthesis well known to those ofskill in the art, for example, by solid phase peptide synthesis methods(e.g., Fmoc synthesis), or by generating a nucleic acid encoding thedesired hybrid protein, for example, by recombinant nucleic acidgeneration methods well known to those of skill in the art (e.g.,restriction cloning, PCR, and/or gene synthesis methods).

In some embodiments, the intein self-excises efficiently only if thefirst residue of the C-terminal extein (e.g., target protein(C) in thestructure described above) is a specific amino acid residue, forexample, a cysteine. In some embodiments, an intein is inserted into atarget protein at a position immediately upstream (N-terminal) of such aresidue (e.g., a cysteine residue). If inserted into such a position,the intein will leave no “scar” in the protein after excision, since theexcision will leave the first extein residue, which, in this case, is aresidue of the native protein. In other embodiments, where no suitableresidue (e.g., a cysteine residue) can be identified for upstream inteininsertion, a sequence comprising the intein domain and an additional,C-terminal amino acid residue (e.g., a C-terminal cysteine residue), maybe inserted into the desired sequence position of the target protein. Insuch cases, intein excision will leave the first extein residue (e.g.,the C-terminal cysteine residue) inserted in the target protein. Thoseof skill in the art will be able to identify suitable residues that canserve as first C-terminal extein residues, and devise strategies forinserting suitable C-terminal amino acid residues with the inteindomains in those embodiments, where suitable first C-terminal exteinresidues are not available in the native target protein sequence.

Any protein can be modified to comprise an intein and, thus, serve as atarget protein. In some embodiments the target protein is a protein theligand-dependent activation of which is useful for a therapeutic,diagnostic, or experimental purpose. In certain embodiments, the targetprotein exhibits an activity in a cell or in a biochemical pathway beingstudied. In other embodiments, the target protein is a therapeuticprotein. In certain embodiments, the target protein is an enzyme (e.g.,an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, ora ligase, such as a kinase, a phosphorylase, a cytochrome p450 enzyme, aprotease, a polymerase, an aldolase, or a phosphatase). In otherembodiments, the target protein is involved in a cell signaling pathway.In certain embodiments, the target protein is a kinase. In certainembodiments, the target protein is a transcription factor. In certainembodiments, the target protein is a transmembrane signaling protein. Insome embodiments, the target protein is a recombinase. In someembodiments, the target protein is an endonucleases, for example, a zincfinger nuclease. In certain embodiments, the target protein is areceptor. In other embodiments, the target protein is a structuralprotein. In some embodiments, the target protein is a reporter protein.In some embodiments, the target protein is a fluorescent protein. Insome embodiments, the target protein is GFP, RFP, BFP, CFP, YFP, or anyenhanced or destabilized variant thereof. In some embodiments, thetarget protein is a mitochondrial protein. In some embodiments, thetarget protein is a fusion of one or more proteins or protein domains.

Typically, the target protein exhibits a biological activity that isdisrupted when the ligand-dependent intein is inserted into the targetprotein. In some embodiments, the resulting hybrid protein does notexhibit the biological activity of the target protein. In someembodiments, the hybrid protein exhibits a diminished level of thebiological activity as compared to the native target protein. In someembodiments, once the intein is excised during protein splicing, thebiological activity is restored in the mature, spliced target protein.In some embodiments, the hybrid protein containing a ligand-dependentintein inserted into the sequence of a target protein is inactive untilit is contacted with an appropriate ligand which binds to the intein isadded. In some embodiments, ligand binding induces the protein splicingactivity of the ligand-dependent intein, which causes the excision ofthe intein and ligation of the resulting ends to form the mature,biologically active target protein.

Intein-Encoding Nucleic Acid Molecules

Some aspects of this invention provide nucleic acid molecules encodingthe ligand-dependent inteins, optimized intein domains, or optimizedligand-binding domains described herein. Such nucleic acid molecules canbe generated for a known intein amino acid sequence by methods wellknown to those of skill in the art, for example, by recombinant DNAtechnology methods (see, e.g., Molecular Cloning: A Laboratory Manual,2nd Ed., Sambrook, Fritsch, and Maniatis (Cold Spring Harbor LaboratoryPress: 1989); Nucleic Acid Hybridization (B. D. Hames & S. J. Higginseds. 1984); Methods in Enzymology (Academic Press, Inc., N. Y.);Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker,Academic Press, London, 1987); Ausubel et al., Current Protocols inMolecular Biology (John Wiley & Sons, Inc., New York, 1999); the entirecontents of each of which are incorporated herein by reference). Incertain embodiments, a nucleic acid molecule encoding a ligand-dependentintein as provided herein is synthesized using a DNA synthesizer. Inother embodiments, the nucleic acid molecule may be excised from avector, for example, a plasmid, or artificial chromosome comprising theintein sequence (e.g., using restriction enzymes). In some embodiments,polymerase chain reaction (PCR) is used to isolate and amplify thedesired sequence. The nucleic acid molecule may also be prepared with amutation in the sequence. In certain embodiments, codon usage may beoptimized for expression in a particular organism, for example, in E.coli, S. cerevisiae, mouse, rat, or human. Methods for generating,deriving, and synthesizing nucleic acid molecules encoding a known aminoacid sequence, and/or optimizing codon usage for an organism ofinterest, are well known to those of skill in the art and the inventionis not limited in this respect.

Cells and Organisms

Some aspects of this invention provide cells comprising a nucleic acidencoding a ligand-dependent intein, a hybrid protein, an intein domain,or a ligand-binding domain, as described herein. In some embodiments,the nucleic acid comprises a sequence encoding a hybrid proteincomprising a target protein of interest and, embedded in the targetprotein sequence, a ligand-dependent intein as provided herein. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis a bacterial cell. In some embodiments, the cell is a yeast cell. Insome embodiments, the cell is a human cell, a mouse cell, a rat cell, ahamster cell, a pig cell, a goat cell, a cow cell, a horse cell, a catcell, or a dog cell. In some embodiments, the cell is an embryonic stemcell. In some embodiments, the cell is a differentiated cell. In someembodiments, the cell is derived from a diseased cell or tissue, forexample, from a malignant cell or tissue.

Some embodiments provide organisms comprising a cell or consisting ofcells comprising an intein, comprising a nucleic acid encoding aligand-dependent intein, a hybrid protein, an intein domain, or aligand-binding domain, as described herein. For example, in someembodiments, a transgenic organism is provided that comprises at leastone cell, in which a nucleic acid molecule encoding a ligand-dependentintein, a hybrid protein, an intein domain, or a ligand-binding domain,as described herein, is integrated into the cellular genome. In someembodiments, the transgene encodes a hybrid protein of a target proteinand a ligand-dependent intein as described herein. In some embodiments,the organism does not express the native target protein in a cell ortissue of interest. For example, in some embodiments, the organism is anorganism in which one or both native alleles of the target protein havebeen rendered non-functional by methods known to those of skill in theart, for example, by knockout, knock-in, mutation, deletion, RNAinterference, or other suitable method. Suitable methods for rendering agenomic allele encoding a target protein of interest non-functional arewell known to those of skill in the art and the invention is not limitedin this respect.

Nucleic acids, for example, nucleic acids encoding a ligand-dependentintein, a hybrid protein, an intein domain, or a ligand-binding domain,as described herein, as well as hybrid proteins comprising an inteininserted into the amino acid sequence of a target protein can beintroduced into a target cell by methods well known to those of skill inthe art, for example, by contacting a target cell with a nucleic acidencoding a hybrid protein (e.g., transfection, transduction, infection,electroporation), or by contacting a target cell with a hybrid proteinfused to a protein transduction domain to effect protein transduction.Such delivery methods are well known in the art and the invention is notlimited in this respect.

Methods of Evolving Ligand-Dependent Inteins

In some embodiments, FACS-based screening methods are provided hereinthat are useful for the directed evolution of inteins for specificapplications, e.g., at a specific temperature range, or in a specificcell or tissue type. In some embodiments, these methods use adiversified intein-library, for example, diversified by error-prone PCRas described in detail elsewhere herein, inserted into a target proteinthat can be detected in a FACS assay, for example, a fluorescentprotein, or a cell-surface protein. In some embodiments, intein activityis coupled to an increase in target protein activity, for example, GFPfluorescence. In some embodiments, a diversified library of inteinsinserted into a detectable target protein is provided and cells arecontacted with this library. Preferably, each cell expresses only onemember of the intein library. In some embodiments, the screening methodcomprises a positive selection, for example, a selection of those cellsexhibiting the highest target protein activity upon exposure to anappropriate ligand. In the case of GFP being used as the target protein,cells exhibiting the highest level of GFP fluorescence after exposure toligand (e.g., 4-HT) are isolated by FACS to obtain a population of cellsexpressing those members of the intein library with the highest splicingactivity. In some embodiments, the screening method further comprises anegative selection. For example, in some embodiments, cells expressinglibrary inteins are subjected to FACS sorting in the absence of ligand.In some embodiments, those cells exhibiting the lowest background targetprotein activity (e.g., the lowest GFP fluorescence) in the absence ofligand are isolated by FACS to obtain a population of cells with thelowest intein activity in the absence of ligand. The positive andnegative selection steps can be combined, in any order, to obtain apopulation of cells expressing library members with the highestligand-dependent splicing activity and the lowest splicing background inthe absence of ligand. In some embodiments, a positive selection step isfollowed by a negative selection step. In other embodiments, a negativeselection step is followed by a positive selection step. In someembodiments, the population if library inteins or the nucleic acidsencoding the library inteins obtained after a positive and a negativeselection step are isolated and subjected to an additional round ofdiversification and selection. FACS selection offers a large dynamicrange that can be exploited for the isolation of active and highlyactive library members, allows analysis of individual library members atthe single-cell level, and supports very high-throughput screens; inthis work, ˜10⁷ cells were screened in a few hours. FACS is also anondestructive method, and cells collected in this manner are robustenough to be cultured in liquid or on solid media immediately followingthe screening process, allowing for multiple rounds of selection beingconducted on a cell population. Further, the ability to culture and thusamplify the cells resulting from each screen simplifies the process ofenriching and isolating desired library members.

Methods of Using Ligand-Dependent Inteins

The ligand-dependent inteins described herein allow for timed modulationof the activity of a target protein of interest in a cell or anorganism, both in vitro or in vivo, by addition or administration of anappropriate ligand. Appropriate ligands are well known to those of skillin the art and some of the ligands described herein, for example,tamoxifen and tamoxifen-analogs (e.g., 4-HT) have been proven non-toxicat effective concentrations in clinical trials. Some appropriate ligandsdescribed herein are used in the clinic for human therapy, and methodsand concentrations for the use of such ligands in vitro, for example, inmammalian cell culture, are well established in the art.

One advantage of the methods described herein is that proteinsplicing-mediated induction of target protein activity exhibits fasterkinetics than modulating transcription or translation of the respectivetarget gene, since the hybrid protein can be activated by inteinexcision without any time-consuming transcription or translation stepinvolved. Accordingly, ligand-dependent inteins as described hereinallow for the study of target proteins in a cell, tissue, organism, orbiological pathway without disturbing transcriptional or translationalpathways. Since the inteins described herein can be induced with a smallmolecule independent of their extein context, they are universallyapplicable, and their use avoids the development of specific inhibitorsof each target protein of interest.

In some embodiments, a hybrid protein, which includes a ligand-dependentintein inserted into a target protein, is used to investigate theactivity of a protein of interest or the role of a protein of interestin vitro or in viva. Ligand-dependent inteins provide a means of rapidlyactivating a protein by the addition of an appropriate ligand, and arethus suitable for investigating biochemical pathways, cell signalingpathways, developmental controls, etc.

A nucleic acid molecule encoding a hybrid protein of a ligand-dependentintein and a target protein as described herein may be transformed intoany cell in which the activity of the target protein is to be assessed.For example, for investigating the role of a particular transcriptionfactor in mammalian cells, a nucleic acid molecule encoding a hybridprotein comprising a ligand-dependent intein described herein insertedinto the amino acid sequence of the transcription factor can be used totransform mammalian cells. In some embodiments, the mammalian cells donot express the native transcription factor, resulting in the onlysource of active transcription factor molecules being splice products ofthe hybrid protein. One of the advantages of the ligand-dependentinteins described herein is that once such an intein or a nucleic acidencoding such an intein is prepared, it may be used in a variety ofextein contexts without further manipulation.

In some embodiments, a hybrid protein comprising a ligand-dependentintein described herein is used for therapeutic purposes. In someembodiments, the hybrid protein or a nucleic acid molecule encoding thehybrid protein is administered to a subject or used to transducer ortransform cells which are subsequently administered to a subject. Incertain embodiments, the hybrid protein is used to treat or prevent aparticular disease in a subject. For example, in some embodiments, anappropriate ligand activating the splicing activity of the intein ispresent only or predominantly in a particular target cell, tissue, ororgan of the subject. Accordingly, hybrid protein splicing an, thus,restoration of target protein activity, only or predominantly takesplace in that cell, tissue, or organ. In some embodiments, aligand-dependent intein as described herein may provide temporal controlover target protein activity. For example, an appropriate ligand, whichactivates the splicing activity of the intein, may be providedexogenously or endogenously at a particular time.

In some embodiments, a hybrid protein comprising an intein as describedherein embedded in the protein sequence of a target protein of interestis expressed in a cell, tissue, or organism. In some such embodiments,it is desirable to eliminate or diminish expression of the native targetprotein in the cell, tissue, or organism. This can be achieved bymethods well known to those of skill in the art, for example, by genetargeting methods. For example, in some embodiments, the native locus ofa gene of interest may be knocked out in a cell, or organism, and anucleic acid molecule encoding a hybrid protein comprising the targetprotein and a ligand-dependent intein as described herein may beintroduced into the cell or organism to obtain a cell or organism inwhich only the ligand-inducible, but not the native target protein isexpressed.

In some embodiments, a method of using a ligand-dependent intein isprovided that involves contacting a target cell with a hybrid proteincomprising a ligand-dependent intein or intein domain, as describedherein, inserted into the amino acid sequence of a target protein ofinterest, or with a polynucleotide encoding such a hybrid protein. Insome embodiments, a method of using a ligand-dependent intein isprovided that involves contacting a cell or tissue comprising orexpressing a ligand-dependent-intein or intein domain, as describedherein, with a ligand binding to the ligand-binding domain of theligand-dependent intein. In some embodiments, the cell or tissue iscontacted with an amount of an appropriate ligand (e.g., 4-HT) thateffects self-excision of the ligand-dependent intein from the hybridprotein in at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or in 100% of the hybrid proteinmolecules. In some embodiments, a tissue is contacted with an amount ofan appropriate ligand (e.g., 4-HT) that effects self-excision of theligand-dependent intein from the hybrid protein in at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or in 100% of the cells in the tissue. In some embodiments,the cell or tissue is contacted with an amount of an appropriate ligand(e.g., 4-HT) that restores an activity of the target protein to at least10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or to at least about 100% of the level of the targetprotein activity measured or expected in a cell or tissue expressing thenative target protein at a similar level as the level of expression ofthe hybrid protein in the target cell or tissue. Methods of contacting acell or tissue in vitro or in vivo with an appropriate ligand and formeasuring self-excision efficiency, levels of hybrid protein, and/or ofmature, spliced target protein, as well as method for measuring thelevel or target protein activity, are described herein. Additionalsuitable methods will be apparent to those of skill in the art and theinvention is not limited in this respect. Suitable methods fordetermining target protein activity will, of course depend on the natureof the target protein and the activity to be measured. Methods formeasuring a variety of target protein activities (e.g., enzymatic,fluorescent, structural, etc.) are well known to those of skill in theart and the invention is not limited in this respect.

In some embodiments, a method of using a ligand-dependent intein isprovided that involves generating an organism, for example, a transgenicorganism, expressing a hybrid protein comprising a ligand-dependentintein or intein domain, as described herein, inserted into the aminoacid sequence of a target protein of interest, in at least one cell. Insome embodiments, a method of using a ligand-dependent intein isprovided that involves administering to the organism comprising orexpressing a ligand-dependent intein or intein domain, as describedherein, a ligand binding to the ligand-binding domain of theligand-dependent intein. In some embodiments, an amount of anappropriate ligand (e.g., 4-HT) is administered that effectsself-excision of the ligand-dependent intein from the hybrid protein inat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or in 100% of the hybrid proteinmolecules. In some embodiments, an amount of an appropriate ligand(e.g., 4-HT) is administered that effects self-excision of theligand-dependent intein from the hybrid protein in at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or in 100% of a cell population, for example, of a cellpopulation in a tissue of interest comprised in the organism. In someembodiments, an amount of an appropriate ligand (e.g., 4-HT) isadministered to the organism that restores an activity of the targetprotein to at least 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or to about 100% of the level ofthe target protein activity measured or expected in a cell, tissue, ororganism expressing the native target protein at a similar level as thelevel of expression of the hybrid protein.

In some embodiments, a method is provided that includes ligand-dependenthybrid protein splicing in a cell, tissue, or organism that is diseasedor serves as a disease model. In some such embodiments, the targetprotein comprised in the hybrid protein exhibits a therapeutic functionupon excision of the ligand-dependent intein from the hybrid protein.

In some embodiments, a method of using an intein described herein isprovided that includes the generation of a hybrid protein comprising amitochondrial target protein and an intein or intein domain as describedherein embedded into the target protein sequence. In some embodiments,the target protein is a highly hydrophobic protein. In some embodiments,the hybrid protein is less hydrophobic than the target protein, based onthe embedded intein or intein domain, allowing for delivery of thehybrid protein to the target organelle and the restoration of targetprotein hydrophobicity and/or other activity in the target organelle(e.g., a mitochondrion).

Additional applications of the ligand-dependent inteins and inteindomains described herein will be apparent to those of skill in the art.The inteins provided herein can be used for any application thatconventional inteins are useful for (e.g., as described in InternationalPCT Patent Application Serial Number PCT/US2005/010805, filed Mar. 30,2005; U.S. Pat. No. 7,192,739, issued Mar. 20, 2007; U.S. Pat. No.7,541,450, issued Jun. 2, 2009; and on pages 1-10, 193-207, 211-229,233-252, and 325-341 of Gross, Belfort, Derbyshire, Stoddard, and Wood(Eds.) Homing Endonucleases and Inteins Springer Verlag Heidelberg, ISBN9783540251064; the contents of each of which are incorporated herein byreference). However, the inteins and intein domains provided herein havethe advantage of optimized efficiency within a temperature range ofabout 30° C. to about 37° C., making them an attractive tool forapplications in mammalian cells.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the example section below.The following examples are intended to illustrate the benefits of thepresent invention and to describe particular embodiments, but do neitherexemplify nor limit the full scope of the invention.

Kits

Some embodiments of this invention provide kits comprising an intein,intein domain, ligand-dependent domain, or hybrid protein as describedherein. In some embodiments, a kit is provided that comprises a nucleicacid molecule encoding an intein, intein domain, ligand-dependentdomain, or hybrid protein as described herein. For example, in somekits, a nucleic acid or a plurality of nucleic acids may be providedthat allow efficient generation of hybrid proteins via restriction orrecombination cloning. For example, some kits may comprise a nucleicacid molecule comprising a sequence encoding a ligand-dependent inteinas provided herein (e.g., as provided in SEQ ID NOs 3-8), wherein theintein-encoding sequence is flanked by multiple cloning sites allowingin-frame cloning of target protein-encoding sequences, target protein(N) and target protein (C), upstream and downstream of theintein-encoding sequence, respectively. In some embodiments, theintein-encoding sequence is comprised in a vector, for example, abacterial plasmid vector, that comprises an origin of replicationallowing for efficient replication and propagation in a bacterial host.In some embodiments, the vector further comprises a mammalian promoterdriving expression of the intein-encoding sequence and any adjacenttarget protein-encoding sequences, in mammalian cells. In someembodiments, the vector further comprises a transcriptional terminationsignal or a transcriptional insulator downstream of the intein-encodingsequence and any adjacent target protein-encoding sequence or cloningsite.

In some embodiments, the kit comprises a container and/or accompanyinginstructions or specifications for the use of the included inteins orintein-encoding. The kit may also include other polynucleotides,vectors, cells, buffers, enzymes, nucleotides, tubes, plates, ligand,maps, sequences, etc. that are useful in connection with theligand-dependent inteins or intein encoding nucleic acids describedherein.

EXAMPLES Materials and Methods Yeast Strains and Media

Media consisted of yeast nitrogen base (Sigma), 4% dextrose, andsynthetic drop out supplements lacking uracil (MP Biomedical). Yeastwere cultured in liquid medium or on agar plates at 30° C. The yeaststrain RDY98 (Erg6del::TRP1 pdr1del::KanMX pdr3::HIS3 ade2-1 trp1-1his3-11,15 ura3-52 leu2-3,112 can1-100) was provided by Professor AllenBuskirk at Brigham Young University. Protein induction was performed inmedia consisting of yeast nitrogen base (Sigma), 4% galactose, 4%raffinose, 0.4% dextrose, synthetic drop out supplements lacking uracil(MP Biomedical), and 1% of 100× penicillin-streptomycin solution(Cellgro) at 30 ° C.

Mammalian Cell Culture

HEK293 cells were cultured in Dulbecco's modified Eagle medium(DMEM):F12 medium with 10% fetal bovine serum (FBS) and 1% of 100×penicillin-streptomycin solution (Cellgro) according to standardprotocols. Transient transfections were performed using Effectene(Qiagen) following the manufacturer's protocol.

Library Construction

Error-prone PCR was carried out using 2-4 and 3-2 intein sequences astemplates using DNA bases 8-oxo-2′-deoxyguanosine (8-oxo-dGTP) and6-(2-deoxy-b-D-ribofuranosyl)-3,4-dihydro-8Hpyrimido-[4,5-C] [1,2]oxazin-7-one (dPTP) purchased from TriLink BioTechnologies as previouslydescribed (Zaccolo et al., 1996) using oligonucleotides 5′-TAT GTA CAGGAA CGC ACT ATA TCT TTC AAA GAT GAC GGG AAC TAC GCA TGC-3′ (SEQ ID NO:14) and 5′-GTG CAC GAC AAC CCC TTC GGC GAC GAG GGT GTG CAG TTC CTC GACCTC GAG-3′ (SEQ ID NO: 15). Mutagenized intein PCR products wereinserted into p416Gall GFP-intein vector pre-cut with Sphl and XhoI (toremove the existing intein sequence) by in vivo homologous recombinationof overlapping PCR fragments as previously described (Raymond et al.,1999).

Plasmid Construction

GFP-intein library members were amplified by PCR from the correspondingp416Ga11 library vector using oligonucleotides 5′-CTC GTT TAGTGA ACC GTCAGA GCC GCC ATG GCA AGC AAA GGA GAA-3′ (SEQ ID NO: 16) and 5′-CTA CTTGTC ATC GTC GTC CTT GTA ATC TTT GTA GAG CTC ATC CAT-3′ (SEQ ID NO: 17).pFLAG-CMV-5.1 (Sigma) was amplified by PCR using oligonucleotides 5′-ACACAT GGC ATG GAT GAG CTC TAC AAA GAT TAC AAG GAC GAC GAT-3′ (SEQ ID NO:18) and 5′-TTC TTC TCC TTT GCT TGC CAT GGC GGCTCT GAC GGTTCA CTA AAC-3′(SEQ ID NO: 19). The PCR products were ligated together throughisothermal assembly (Gibson et al., 2009). The resulting ligated vectorswere purified using Min-Elute columns (Qiagen) and eluted with 10 μLdeionized water. 1 μL of this elution was transformed into NEB Turbochemically competent E. coli cells (New England Biolabs) and plated ontoLB+carbenicillin agar plates. Plates were incubated overnight at 37° C.,and individual colonies were picked and sequenced to verify correctplasmid construction.

p3XFlag-CMV-14 (Sigma) vectors with Gli1-intein (2-4 and 3-2), andGli3T-intein (2-4 and 3-2) sequences were previously described (Yuen etal., 2006). p3XFlag-CMV-14 mCherry-intein (2-4 and 3-2) vectors wereconstructed by cloning the 2-4 and 3-2 intein sequences into the pRSET-BmCherry vector provided by Professor Roger Tsien (University ofCalifornia at San Diego). The mCherry-intein 2-4 and mCherry-intein 3-2sequences were then amplified using oligonucleotides introducing a 5′EcoRI site and a 3′ BamHI site and ligated into EcoRI- andBamHI-digested p3XFlag- CMV-14 vector. 30R3-1 intein sequence and 37R3-2intein sequence were amplified using the following oligonucleotidescompatible with mCherry, Gli1, or Gli3 contexts.

mCherry: 5′-TTC GAG GAC GGC GGC GTG GTG ACC GTG TGC CTT GCC GAG GGTACC-3′ (SEQ ID NO: 20) and 5′-GCC GTC CTG CAG GGA GGA GTC CTG GCA GTTGTG CAC GAC AAC CCC-3′ (SEQ ID NO: 21). Gli1:5′-ATC CAC GGG GAG CGG AAGGAA TTC GTG TGC CTT GCC GAG GGT ACC-3′ (SEQ ID NO: 22) and 5′-CTC CCTCGA GCA ACC TCC CCA ATG GCA GTT GTG CAC GAC AAC CCC-3′ (SEQ ID NO: 23).Gli3:5′-ATT CAT GGA GAA AAG AAG GAA TTC GTG TGC CTT GCC GAG GGT ACC-3′(SEQ ID NO: 24) and 5′-CTC TCG AGA ACA ATC AAG CCA GCG GCA GTT GTG CACGAC AAC CCC-3′ (SEQ ID NO: 25). The p3XFlag-pCMV-14 vector was amplifiedusing oligonucleotides 5′-CTC GTC GCC GAA GGG GTT GTC-3′ (SEQ ID NO: 26)and 5′-ATC GAA GAT TCG GGT ACC CTC-3′ (SEQ ID NO: 27). The intein PCRproducts and the vector PCR product were ligated together through theisothermal assembly method (Gibson et al., 2009) and the resultingligated material was treated as discussed above.

FACS Screening and Analysis

Yeast cells transformed with library plasmids were cultured for 24 hrsin the appropriate synthetic drop out media in 30° C. Cells were washedand resuspended in protein induction media and cultured for another 24hrs at 30° C. After 24 hrs of protein induction, cells were treated with1 μM 4-HT or left untreated with 4-HT as appropriate for the prescribedamount of time in either 30° C. or 37° C. After the appropriate lengthof time, cells were harvested by washing once in PBS, then resuspendedin PBS with 0.1% bovine serum albumin (BSA) (Sigma). Cell sorting wasperformed using a MoFlo cell sorter (DakoCytomation). Cell fluorescenceanalysis was carried out on a BD LSRII cell analyzer.

HEK293 cells were grown in 10 cm dishes or 6-well plates and transfectedwith relevant mammalian vectors using Effectene. After growth in theabsence of 4-HT or in the presence of 1 μM 4-HT for 24 hours, cells weretrypsinized and resuspended in 500 μL of phosphate buffered saline with1% FBS and 75 U/mL DNase (New England Biolabs). Cell fluorescenceanalysis was carried out on a BD LSRII cell analyzer.

Western Blots

Western blots were performed using Nu-PAGE 12% Bis-Tris gels(Invitrogen) in 3-(Nmorpholino) propanesulfonic acid (MOPS)-sodiumdodecyl sulfate (SDS) buffer (Invitrogen). SDSpolyacrylamide gelelectrophoresis (PAGE) and Western blotting were performed usingstandard protocols. Gels were transferred onto polyvinylidene fluoride(PDVF) membranes (Millipore). Western blots were processed using a mouseanti-FLAG antibody (Sigma) as the primary antibody and a secondary AlexaFluor 800-conjugated goat anti-mouse antibody (Li-cor Biosciences), thenvisualized and quantitated using an Odyssey imager (Li-cor Biosciences).

Reversion Mutant Construction

Each evolved amino acid change was the result of a single nucleotidemutation. Each reversion mutant was generated using the QuikChangemethod (Stratagene) with Pfu Turbo and the following oligonucleotides(the mutated base pair is underlined in each oligonucleotide pair).

A34V: (SEQ ID NO: 28) 5′-CGC AAG CCT ATT CAT GTC GTG GCT GTT GCC AAG GACGGA ACG CTG CTC GCG-3′ and (SEQ ID NO: 29)5′-CGC GAG CAG CGT TCC GTC CTT GGC AAC AGC CAC GACATG AAT AGG CTT GCG-3′. T66I: (SEQ ID NO: 30)5′-GGG TTG CGG ATC GCC GGT GGC GCC ATC GTG TGG GCGACA CCC GAT CAC AAG-3′ and (SEQ ID NO: 31)5′-CTT GTG ATC GGG TGT CGC CCA CAC GAT GGC GCC ACCGGC GAT CCG CAA CCC-3′. P124L: (SEQ ID NO: 32)5′-TTG TTG GAT GCT GAG CCC CCC ATA CTC TAT TCC GAGTAT GAT CCT ACC AGT-3′ and (SEQ ID NO: 33)5′-ACT GGT AGG ATC ATA CTC GGA ATA GAG TAT GGG GGGCTC AGC ATC CAA CAA-3′. C178R: (SEQ ID NO: 34) 5′-CCA TGA TCA GGC CCACCT TCT AGA ACG TGC CTG GCT AGA GAT CCT GAT GAT-3′. and (SEQ ID NO: 35)5′-ATC ATC AGG ATC TCT AGC CAG GCA CGT TCT AGA AGGTGG GCC TGA TCA TGG-3′. K328T: (SEQ ID NO: 36)5′-GAG CAT CTG TAC AGC ATG AAG TAC ACG AAC GTG GTGCCC CTC TAT GAC CTG-3′ and (SEQ ID NO: 37)5′-CAG GTC ATA GAG GGG CAC CAC GTT CGT GTA CTT CATGCT GTA CAG ATG CTC-3′. G375E: (SEQ ID NO: 38)5′-TTC CTG CAC GAC ATG CTG GCG GAA GAA CTC CGC TATTCC GTG ATC CGA GAA-3′ and (SEQ ID NO: 39)5′-TTC TCG GAT CAC GGA ATA GCG GAG TTC TTC CGC CAGCAT GTC GTG CAG GAA-3′.

Results Evolution Scheme for Improved 4-HT-Dependent Inteins

To improve the splicing characteristics of the evolved 4-HT dependentinteins, the high-throughput fluorescence-activated cell sorting (FACS)screen previously used to isolate active and inactive inteins from mixedstarting populations (Buskirk et al., 2004) was modified (FIG. 1A). The4-HT-dependent intein was genetically inserted in place of Cys 108 ofGFP(uv), a FACS-optimized GFP mutant, which places the intein near themid-point of a β-strand and abolishes fluorescence until splicing takesplace (Ormo et al., 1996; Buskirk et al., 2004). During positive screensfor intein splicing activity, cells that exhibited GFP fluorescence inthe presence of 4-HT were collected, while during negative screens cellsthat remained non-fluorescent in the absence of 4-HT were collected(FIG. 1B). Using error-prone PCR with mutagenic dNTPs (Zaccolo et al.,1996), point mutations were randomly introduced into the genes of thetwo best inteins (including the ligand-binding domain) resulting from aprevious intein evolution effort, the 2-4 and 3-2 inteins (described indetail in International Patent Application Serial NumberPCT/US2005/010805, filed Mar. 30, 2005; U.S. Pat. Ser. No. 7,192,739,filed Mar. 30, 2005; and U.S. Pat. Ser. No. 7,541,450, filed Mar. 19,2007; the entire contents of each of which are incorporated by referenceherein). The resulting intein gene library was cloned into the p416Gallvector in S. cerevisiae RDY98 using gap repair homologous recombination(Raymond et al., 1999) to obtain a starting library size of 7×10₆clones. This starting library was subjected to two evolution efforts inparallel, one conducted at 30° C. and one at 37° C. (FIG. 1C).

Each round of evolution consisted of at least two positive screens andone negative screen (FIG. 1B). Positive screen 1 (P1) for each roundcollected the 5% most fluorescent library members in the presence of4-HT. The second positive screen (P2) in each round collected librarymembers that exhibited better splicing activity than the parental 3-2intein in the presence of 4-HT by collecting cells that were morefluorescent than cells transformed with a 3-2 intein-GFP construct. InRound 2, a third positive screen was carried out (P3) that furtherenriched for library members with better splicing activity than the 3-2intein in the presence of 4-HT. As the final screening step in eachround of evolution, a single negative screen (N) collected librarymembers that did not generate spliced GFP (i.e., were not fluorescent)in the absence of 4-HT. Surviving gene pools were diversified after eachround. Following Round 1, the genes of the surviving library members ineach of the two libraries were separately mutagenized using error-pronePCR before re-cloning into yeast as the starting library for Round 2.After Round 2, surviving genes from the 30° C. and 37° C. screens werecombined and subjected to in vitro homologous recombination using theStEP method (Zhao and Zha, 2006). The resulting recombined library wassubjected to separate Round 3 screens at 30° C. and at 37° C. Overall,the entire evolution process comprised three complete rounds containing10 individual screening steps each for the 30° C. and the 37° C. efforts(FIG. 1C).

Splicing Characteristics of Evolved Inteins

Clones from the 30° C. and the 37° C. libraries surviving each of therounds of evolution were isolated and the genes encoding their inteinswere sequenced. Three sequences each from the 30° C. and 37° C.libraries following Round 3 were selected for detailed characterizationon the basis of their high degree of abundance in the final evolvedpools. These six intein sequences are summarized in Table 1. The newlyevolved clones are designated 30RX-Y (from evolution at 30° C.) or37RX-Y (from evolution at 37° C.), where X refers to the round numberfrom which the clone was isolated and Y refers to the clone numberwithin that round. Mutations Va134Ala, Ile66Thr, Thr328Lys, andGlu375Gly are shared among clones in both the 30° C. and 37° C.libraries. Leu124Pro was observed only in the 30° C. library, andAsp129Asn and Cys178Arg are only observed in the 37° C. library.

TABLE 1 Mutations isolated in evolved inteins. Three clones each fromthe 30° C. and 37° C. evolution efforts were chosen based on theirabundance among DNA sequences surviving Round 3. The mutations comparedwith the 3-2 intein sequence are shown. Val34 Ala, Ile66Thr, andGlu375Gly are mutations in the interin, whereas Leu124Pro, Asp129Asn,Cys178Arg, and Thr328Lys are mutations in the ligand-binding domain.evolved intein clone intein mutations ligand-binding domain mutationsintein mutation 30R3-1 Val34Ala Ile66Thr Leu124Pro Thr328Lys Glu375Gly30R3-2 Val34Ala Ile66Thr Thr328Lys 30R3-3 Val34Ala Ile66Thr Leu124ProThr328Lys 37R3-1 Val34Ala Ile66Thr Asp129Asn Cys178Arg Thr328LysGlu375Gly 37R3-2 Val34Ala Ile66Thr Cys178Arg Thr328Lys 37R3-3 Ile66ThrCys178Arg Thr328Lys

Amino acid sequences of inteins 2-4, and 3-2 are given below. The finalamino acid in each of these sequences is an appended cysteine, oftenreferred to as the first amino acid of the C-terminal extein in theliterature. Bold and underlined residues constitute the N-terminal andC-terminal intein domains, derived from residues 1-94 and 383-440 ofRecA, respectively. The two 6 amino acid linkers are in italics. Thebold residues in the center are derived from residues 304-551 of thehuman estrogen receptor and comprise the ligand-binding domain of theseligand-dependent inteins.

2-4 intein: (SEQ ID NO: 1)CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDR VA GPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHA GGSGAS RVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC 3-2 intein: (SEQ ID NO: 2)CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDR VA GPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHA GGSGAS RVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC

The amino acid sequences of six evolved inteins are provided below.Amino acid changes relative to the 3-2 parent intein are underlined.

30R3-1 intein: (SEQ ID NO: 3)CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC 30R3-2 intein:(SEQ ID NO: 4) CLAEGTRIFDPVTGITHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC 30R3-3 intein:(SEQ ID NO: 5) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC 37R3-1 intein:(SEQ ID NO: 6) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC 37R3-2 intein:(SEQ ID NO: 7) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC 37R3-3 intein:(SEQ ID NO: 8) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHNC

All six evolved inteins were assayed for splicing function in the GFPcontext in yeast cells at both 37° C. and 30° C., and their activitieswere compared with those of the original 2-4 and 3-2 inteins under thesame conditions. A FLAG-tag was appended at the C-terminal end of theGFP-intein sequence to facilitate detection of both the spliced andunspliced protein products. Cells treated with 1 μM 4-HT or without 4-HTat time points from 1 hour to 24 hours were subjected to FACS analysis(FIG. 6), and the spliced and unspliced proteins in the correspondingcell lysates were quantified by Western blot (FIG. 2A) and densitometry.The percentage of spliced protein was calculated as the amount ofspliced protein divided by the total amount of spliced+unspliced proteinfor each sample.

In yeast cells at 37° C., all six newly evolved inteins exhibitsubstantially faster production of spliced proteins as well as asignificantly higher percentage of spliced product in the presence of4-HT as compared to the parental 2-4 and 3-2 inteins (FIG. 2B). Comparedto the original 2-4 or 3-2 inteins, the most active inteins at 37° C.generate up to 8-fold more spliced protein 3 hours after 4-HT additionand up to 5-fold more spliced protein at 6 hours after 4-HT addition(FIG. 2B). For example, while the 2-4 and 3-2 inteins generated 17-27%spliced GFP after 6 hours at 37° C., the six newly evolved inteinsresulted in 70-86% spliced GFP at the same time point. Likewise, whilethe 2-4 and 3-2 inteins did not generate significant amounts of splicedprotein 1 hour after 4-HT treatment, the newly evolved inteins produced25-35% spliced protein at this early time point. For five of the sixclones (all but clone 30R3-3); splicing in the absence of 4-HT remainedlow (typically<10% after 24 hours; see FIG. 2C). Total proteinexpression per yeast cell of all six inteins at each time point werewithin 10% of the expression levels observed with the 3-2 intein(unpublished data), suggesting that the newly evolved inteins do notalter protein expression levels compared with the 3-2 intein and are notunusually susceptible to degradation.

In yeast cells grown at 30° C., five of the six newly evolved inteins(all but clone 37R3-1) exhibit more efficient splicing by the six-hourtime point compared to the 2-4 and 3-2 inteins (FIG. 2D). Splicing inthe absence of 4-HT after 24 hours was generally≦15%, the level ofbackground splicing observed for the 3-2 intein after 24 hours (FIG.2E). The newly evolved inteins generated 3.6- to 7-fold higher levels ofspliced protein at 3 hours, and 1.6- to 2.6-fold higher levels ofspliced protein at 6 hours, relative to the 3-2 or 2-4 inteins,respectively. At later time points (12 or 24 hours) splicingefficiencies for the newly evolved inteins excluding 37R3-1 weregenerally high (≧70%), similar to that of the 3-2 intein. As observed inthe 37° C. assays, the protein expression levels per yeast cell of eachintein were within ±10% of the level observed with the 3-2 intein ateach of the time points. Taking into consideration splicing rate,overall splicing efficiency in the presence of 4-HT, and backgroundsplicing in the absence of 4-HT, the best performing evolved inteinclone for use at 37° C. was 37R3-2, and the best clone at 30° C. was30R3-1. Together, these results indicate that the evolution strategydescribed above resulted in inteins with substantially improved splicingspeed and yields of spliced protein in yeast at 30° C. and especially at37° C., without significantly impairing 4-HT dependence.

Ligand-Dependent Splicing of Newly Evolved Inteins in Mammalian Cells

All six evolved intein sequences described above in the GFP context werecloned into a pCMV promoter-based mammalian expression vector with aC-terminal FLAG-tag for Western blot analysis. HEK293 cells weretransfected with these vectors, incubated for 24 hours at 37° C., thentreated with Dulbecco's modified Eagle medium (DMEM):F12 with 10% fetalbovine serum (FBS) containing 1 μM final concentration of 4-HT or withthe same medium lacking 4-HT. The cells were incubated at 37° C. for anadditional 12-24 hours then harvested for FACS (FIG. 7) and Western blotanalyses (FIG. 3A). Consistent with the characteristics of the newlyevolved inteins in yeast cells, the three assayed clones evolved at 37°C. all exhibit faster GFP splicing kinetics and higher overall splicingyields at both the 12-hour and 24-hour time points compared with the 2-4or 3-2 inteins (FIG. 3B). The best 37° C. clone, 37R3-2, exhibited3.8-fold and 2.2-fold higher GFP splicing efficiency after 24 hours thanthe original 2-4 and 3-2 intein, respectively (73% spliced GFP for37R3-2 vs. 19% for 2-4 and 33% for 3-2). Background splicing in theabsence of 4-HT was not observed for 37R3-2 (FIG. 3B), furtherconsistent with the high ratio of ligand-induced splicing to backgroundsplicing of 37R3-2 observed in yeast cells. The inteins evolved at 30°C. also exhibit similarly improved (˜2- to 6-fold) splicing kinetics andsplicing efficiencies at 12 and 24 hours relative to that of inteins 2-4and 3-2 (FIG. 3C). The best 30° C. library clone (30R3-1) generated 72%spliced GFP after 24 hours, compared with 33% and 19% spliced GFP forthe 3-2 and 2-4 inteins, respectively, while splicing with≦3% efficiencyin the absence of 4-HT (FIG. 3C).

Interestingly, the fraction of protein splicing that was completed by 12hours relative to an endpoint of 24 hours was also greater for the newlyevolved inteins compared with the 2-4 and 3-2 inteins. For example, anaverage of 59% of the total amount of spliced GFP in mammalian cellsafter 24 hours was present after 12 hours among the three 37° C. libraryclones, and an average of 57% of the total amount of spliced GFP after24 hours was present after 12 hours among the three 30° C. libraryclones. For comparison, 45% and 31% of the total spliced GFP after 24hours was present at 12 hours for the 3-2 and 2-4 inteins, respectively.These results collectively indicate that in live mammalian cells at 37°C. the newly evolved inteins exhibit increased splicing rate and higherextent of splicing compared with the original evolved inteins, whilemaintaining low background splicing in the absence of 4-HT.

Evolved Intein Properties in Different Proteins in Mammalian Cells

The generality of 4-HT-dependent splicing of the two best evolvedinteins (30R3-1 and 37R3-2) was investigated by inserting these inteinsinto three other protein contexts in addition to GFP in mammalian cells.The inteins were inserted into mCherry, a red fluorescent protein, inplace of Thr 113, the residue in mCherry that corresponds to the Cysresidue used for intein insertion in GFP. As in the case of GFP, thisplacement positions the intein near the mid-point of a β-strand andabolishes mCherry fluorescence until splicing takes place (vide infra).The corresponding genes were introduced into HEK293 cells as describedabove for the GFP-intein genes, then treated with media containing 1 μM4-HT or with media lacking 4-HT. These cells were incubated at 37° C.for an additional 12 to 24 hours, then harvested for FACS (FIG. 8) andWestern blot analysis.

Both of the 37R3-2 and the 30R3-1 evolved inteins continued to exhibitsignificant improvement in splicing performance over the 2-4 and 3-2inteins in the context of mCherry (FIG. 4A). The 37R3-2 intein resultedin 72% spliced mCherry protein after 24 hours and 43% spliced proteinafter 12 hours. The 30R3-1 intein resulted in 54% and 33% splicedmCherry after 24 and 12 hours, respectively. Thus the percentage ofspliced mCherry generated by the 37R3-2 intein at 24 hours or 12 hourswas ˜3-fold higher than that of 3-2 intein, and ˜5-fold higher than thatof the 2-4 intein. Background splicing in the absence of 4-HT was ≦3%for all inteins assayed in this context.

The splicing characteristics of the newly evolved inteins wasinvestigated in the contexts of two additional mammalian proteins, Gli1and Gli3T. Gli1 and Gli3 are transcription factors that mediate Hedgehogsignaling (Koebemick and Pieler, 2002) and are important in many keydevelopmental processes such as spinal cord patterning (Bai et al.,2004) and limb development (Barna et al., 2005). Gli3T is a C-terminallytruncated form of the transcription factor Gli3 that is used as atranscriptional repressor (Wang et al., 2000). Gli1 and Gli3T are largeproteins, 122 kDa and 85 kDa respectively, and are both structurallyunrelated to GFP, mCherry, and structurally distinct from each other.The 37R3-2 and 30R3-1 inteins were inserted genetically in place of Cys273 of the Gli1 protein and in place of Cys 515 of the Gli3T protein asdescribed previously (Yuen et al., 2006). The insertion of inteins intothese proteins at these positions abolishes their activities untilsplicing takes place (Yuen et al., 2006). The resulting constructs wereintroduced into HEK293 cells and splicing was evaluated by Western blotas described above.

Consistent with their enhanced performance characteristics in the GFPand mCherry contexts, the newly evolved inteins in Gli1 and in Gli3Tresulted in significantly higher (˜2- to 4-fold in Gli1, and ˜3- to8-fold in Gli3T) percentages of spliced protein compared with the 3-2 or2-4 inteins (FIGS. 4B and 4C). Up to 48% and 60% of Gli1 protein wasspliced by the newly evolved inteins after 12 and 24 hours,respectively, while the previously evolved 3-2 intein resulted in 22%and 32% splicing under the same conditions. Likewise, the newly evolvedinteins generated up to 43% and 51% spliced Gli3T protein after 12 and24 hours, compared with 10% and 18% for the 3-2 intein.

It was previously noted (Yuen et al., 2006; unpublished data) that the3-2 intein undergoes background splicing in the absence of 4-HT to agreater extent than the 2-4 intein, and the Gli1 and Gli3T data in FIGS.4B and 4C replicated these observations. The background splicing ofnewly evolved clone 37R3-2 across all four proteins tested (GFP,mCherry, Gli1, and Gli3T) is very low and often was not detectable.Clone 30R3-1 in general resulted in a slightly higher degree ofbackground splicing, but this level of splicing in the absence of 4-HTremained ≦3% in all four proteins tested in this work, and generally wassimilar to or slightly lower than the background splicing of the 3-2intein. Taken together, these results establish the overall superiorsplicing kinetics and splicing efficiency without significant backgroundsplicing for both newly evolved inteins 37R3-2 and 30R3-1 in a varietyof protein contexts in mammalian cells.

Mutational Analysis of Evolved Inteins 30R3-1 and 37R3-2

To probe which mutations were responsible for the improved properties ofevolved inteins 30R3-1 and 37R3-2, each of the mutations (five in 30R3-1and four in 37R3-2) was systematically reverted to the correspondingamino acid present in the original 3-2 intein. Each of these reversionmutants was inserted genetically into GFP and transformed into RDY98yeast cells. Protein expression was induced for 24 hours at 30° C. andthe resulting cells were incubated for six hours in the presence orabsence of 4-HT at 30° C. and 37° C. Protein splicing was assessed byFACS and Western blot analysis as described above.

Compared with the 3-2 intein, both newly evolved intein variants containVal34Ala and Thr328Lys mutations. These two changes also correspond tothe two differences between the 2-4 intein and the 3-2 intein, whichevolved from the 2-4 intein. In both newly evolved inteins the aminoacids at these two positions were the residues present in the 2-4intein. Consistent with the lower splicing activity in the absence orpresence of 4-HT of the 2-4 intein relatively to the 3-2 intein, thereversion of Ala34 back to Val and Lys328 back to Thr resulted insignificantly higher background splicing, especially in the 37R3-2intein, together with slightly higher splicing efficiency in thepresence of 4-HT (FIGS. 5A-5D). These results suggest that residues 34and 328 in the newly evolved inteins modestly modulate splicing activityin a ligand-independent manner, and that the presence of Ala34 andLys328 serves to decrease background splicing by a significant fraction(˜2- to 4-fold), while lowering splicing efficiency in the presence of4-HT by a much smaller relative fraction (˜10% lower).

The Glu375Gly mutation was also present in both the 30R3-1 and the37R3-2 inteins. Reversion of this mutation resulted in substantiallyincreased (2- to 10-fold) background splicing in the absence of 4-HTwithout any significant change in splicing efficiency in the presence of4-HT (FIGS. 5A-5D). This mutation therefore likely serves to suppresssplicing activity in a manner that is selective for the conformation ofthe ligand-free intein.

Among the other two mutations in 30R3-1, neither was present in 37R3-2.Reverting the Thr at residue 66 to Ile in 30R3-1 resulted in nosignificant change in intein activity other than a slight decrease in4-HT-triggered splicing at 30° C. (FIG. 5A). Likewise, reversion ofPro124 to Leu also resulted in similar splicing activities in thepresence or absence of 4-HT as the 30R3-1 intein (FIGS. 5A and 5B).These results suggest that Thr66 and Pro124 may not contribute to theobserved changes in splicing activity, or may only contribute toimproved splicing in combination with one or more additional mutations.

The Cys178Arg mutation is the sole change in 37R3-2 that is not presentin 30R3-1. Reversion of this mutation modestly decreases splicingefficiency in the presence of 4-HT and may also slightly increasebackground splicing (FIGS. 5C and 5D). Interestingly, these resultssuggest that no single mutation in the 30R3-1 or 37R3-2 inteins isresponsible for the substantial majority of the observed ˜2- to 5-foldimproved splicing of GFP in yeast at 30° C. or 37° C. compared with theparental 2-4 or 3-2 inteins. Instead, the observations described hereinsuggest that the combination of four or five mutations, each of whichcontribute modest improvements, cumulatively result in the substantiallyfaster and more efficient splicing in the presence of 4-HT whilepreserving or decreasing the extent of background splicing relative tothe 3-2 intein.

Discussion

The intein evolution efforts described here were performed under thehypothesis that inteins evolved under more stringent selectionconditions and at 37° C. may yield ligand-dependent inteins withsuperior splicing yields, faster splicing kinetics, and/or lowerbackground splicing than those previously reported. Parallel 37° C. and30° C. screening conditions were used to explore a wider range ofpossible advantageous mutations than might have been surveyed through37° C. screens alone. Indeed, mutations in the two evolved inteins withthe best overall properties, clones 30R3-1 and 37R3-2, arose from boththe 30° C. and 37° C. libraries. These two inteins in the presence of4-HT exhibited substantially higher yields of spliced protein and fastersplicing, while maintaining comparable or slightly improved (decreased)amounts of background splicing in the absence of 4-HT.

It is interesting that the newly evolved inteins exhibited fastersplicing kinetics (i.e., reached a higher percentage of final splicedprotein levels at early time points) compared with the parental inteinseven though the methods used in this work did not explicitly screen forimproved splicing kinetics. It is possible that some of the mutationsdiscovered in this work improve the kinetics of the splicing reactionitself, but it is equally likely that these mutations increased the rateof other steps in the ligand-induced splicing process such as proteinfolding, binding or dissociation from Hsp90 or other proteins (Feil etal., 1996; Kellendonk et al., 1996; Zhang et al., 1996; Danielian etal., 1998; Picard et al., 2000; Buskirk et al., 2004; Yuen et al.,2006), or conformational changes that influence the ability of theintein to undergo splicing.

The FACS-based screening method used here, in which intein activity iscoupled to an increase in GFP fluorescence, is ideally suited for thistype of laboratory evolution in which starting proteins possessingdetectable activities are evolved to higher levels of activity underspecific sets of conditions. FACS offers a large dynamic range that iscrucial for distinguishing active and highly active library members,allows analysis of individual library members at the single-cell level,and supports very high-throughput screens; in this work, ˜10⁷ cells werescreened in a few hours. FACS is also a nondestructive method, and yeastcells collected in this manner are robust enough to be cultured inliquid or on solid media immediately following the screening process.The ability to culture and thus amplify the cells resulting from eachscreen simplifies the process of enriching desired library members.These features together enabled improved variants to emerge by tuningthe screen to capture progressively more fluorescent cells withprogressively higher intein activity levels.

The use of small molecules to modulate protein structurepost-translationally in living cells remains an attractive approach tostudying protein function. The ligand-dependent intein, like otherpost-translationally triggered protein manipulation methods (Stankunaset al., 2003; Wang et al., 2003; Bayle et al., 2006), facilitatestemporal control of protein structure as well as dose-dependenttitration of spliced protein levels (Buskirk et al., 2004; Yuen et al.,2006). Inteins offer some features that may make them particularly wellsuited for certain applications, especially given the improvements insplicing characteristics described herein. While traditional chemicalgenetic approaches require the discovery of small molecules that perturbthe activity of each protein of interest, ligand-dependent inteinsconfer dependence on a single small molecule (e.g., 4-HT) on a varietyof proteins of interest with single-target specificity. The proteinsplicing process leaves behind only a single Cys residue, or no scar incases in which the target protein naturally contains a Cys residue in alocation that results in loss of protein function upon intein insertion.Moreover, small-molecule-triggered protein splicing ispseudo-autocatalytic and does not require additional cellular componentsor specific conditions that may not be easy to establish for someintracellular proteins.

The small-molecule-dependent inteins developed here may be particularlysuited for studying signaling pathways because of the minimal cellularperturbations required to achieve control over protein function. The useof the evolved inteins does not require changes to regulatory regions ofgenes and does not require the expression of any other proteins ornucleic acids. Since cell disruption is minimized, proteins that are apart of complex mammalian signaling pathways—for example those in whichfeedback regulation plays a significant role—have a greater chance ofmaintaining their native regulatory networks. Further, thedose-dependent nature of ligand-dependent intein-splicing allows for thefine control of functional protein levels.

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All publications, patents and sequence database entries mentionedherein, including those listed in the Summary, Detailed Description,Examples, and References sections above, are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

It is to be understood that the invention encompasses all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of theclaims or from relevant portions of the description is introduced intoanother claim. For example, any claim that is dependent on another claimcan be modified to include one or more limitations found in any otherclaim that is dependent on the same base claim.

Where the claims recite a composition, it is to be understood thatmethods of using the composition for any of the purposes disclosedherein are included, and methods of making the composition according toany of the methods of making disclosed herein or other methods known inthe art are included, unless otherwise indicated or unless it would beevident to one of ordinary skill in the art that a contradiction orinconsistency would arise.

Where ranges are given, endpoints are included, and, if not otherwiseindicated or inconsistent, values that are expressed as ranges canassume any specific value or subrange within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

1-29. (canceled)
 30. An estrogen-binding domain comprising the aminoacid sequenceNSLALSLTADQMVSALLDAEPPIL*YSEYD*PTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLEC*AWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYT*NVVPLYDLLLEM LDAHRLHA(SEQ ID NO: 13), wherein at least one of the residues L*, D*, C*, or T*is mutated.
 31. The estrogen-binding domain of claim 30, wherein theestrogen-binding domain comprises at least one of the followingmutations: L*P, D*N, C*R, or T*K.
 32. The estrogen-binding domain ofclaim 30, wherein the estrogen-binding domain is fused to an N-terminalintein domain and a C-terminal intein domain, forming a ligand-dependentintein.
 33. The estrogen-binding domain of claim 30, wherein theN-terminal intein domain and the C-terminal intein domain areRecA-derived intein domains.
 34. The estrogen-binding domain of claim33, wherein the N-terminal intein domain comprises the sequenceCLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAV*AKDGTLLARPVVSWFDQGTRDVIGLRIAGGAI*VWATPDHKVLTEYGWRAAGELRKGDRVA (SEQ ID NO: 10); and/or theN-terminal intein domain comprises the sequenceRVQAFADALDDKFLHDMLAEE*LRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN (SEQ ID NO:11).
 35. The estrogen-binding domain of claim 34, wherein at least oneof amino acid residues V*, I*, or E* are mutated.
 36. Theestrogen-binding domain of claim 35, wherein the intein comprises atleast one of the following mutations: V*A, I*T, or E*G. 37-62.(canceled)